Compositions, kits, and methods for the modulation of immune responses using galectin-1

ABSTRACT

The present invention is based, in part, on the discovery that galectin-1 (Gal1) plays a role in immune disorders, including Hodgkin lymphoma. Accordingly, the invention relates to compositions, kits, and methods for detecting, characterizing, modulating, preventing, and treating immune disorders, e.g., Hodgkin lymphoma.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/959,830, filed on Jul. 17, 2007; the entire contents of theapplication is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Classical Hodgkin lymphoma (cHL) is a B-cell malignancy diagnosed inapproximately 20,000 new patients in North America and Europe each year;over 90% of these patients are young adults. Classical HLs include smallnumbers of malignant Reed-Sternberg (RS) cells within an extensiveinflammatory infiltrate (Re et al. (2005) J Clin Oncol 23:6379-6386)which includes abundant T helper (Th)-2 and T regulatory (T_(reg))cells. The tumor cells derive from pre-apoptotic germinal center B cellsthat have undergone crippling mutations of their rearrangedimmunoglobulin genes (Re et al. (2005) J Clin Oncol 23:6379-6386;Kanzler et al. (1996) J Exp Med 184:1495-1505). Classical HL RS cellslack B-cell receptor-mediated signals and rely on alternative survivaland proliferation pathways activated by transcription factors such asNF-κB and AP1 (Mathas et al. (2002) EMBO J. 21: 4104-4113; Kuppers etal. (2002) Ann Oncol 13:11-18; Schwering et al. (2003) Blood101:1505-1512). In cHL, the tumor cells exhibit constitutive AP1activation, express high levels of the AP1 components, cJUN and JUNB,and depend upon AP1-mediated proliferation signals (Mathas et al. (2002)EMBO J. 21: 4104-4113).

Although primary cHLs have a brisk inflammatory infiltrate, there islittle evidence of an effective host anti-tumor immune response. Thereactive T-cell population includes predominantly Th2-type and CD4⁺CD25^(high) FOXP3⁺ T_(reg) cells that directly suppress immune responsesand protect cHL RS cells from immune attack (Re et al. (2005) J ClinOncol 23:6379-6386; Marshall et al. (2004) Blood 103:1755-1762; Gandhiet al. (2006) Blood 108:2280-2289, Ishida et al. (2006) Cancer Res66:5716-5722); Th1, NK and cytotoxic T cells are markedlyunder-represented. In addition, primary cHLs are characterized by aunique cytokine and chemokine profile, including IL-4, IL-5, IL-10 andIL-13 (Re et al. (2005) J Clin Oncol 23:6379-6386; Skinnider et al.(2002) Leuk Lymphoma 43:1203-1210). In fact, IL-13 is a critical growthfactor for cHL RS cells (Re et al. (2005) J Clin Oncol 23:6379-6386;Skinnider et al. (2002) Leuk Lymphoma 43:1203-1210). However, themolecular signals and endogenous factors responsible for creating andmaintaining the Th2-skewed immunosuppressive microenvironment in cHLremain to be defined.

Galectins have recently emerged as novel regulators of immune cellhomeostasis, and tumor immune escape (Rabinovich et al. (2002) TrendsImmunol 23:313-320; Liu and Rabinovich (2005) Nature Reviews Cancer5:29-41; Rubinstein et al. (2004) Cancer Cell 5:241-251; Le et al.(2005) J Clin Oncol 23:8932-8941). Galectin-1 (Gal1), an evolutionarilyconserved member of this family (Vasta et al. (2004) Curr Opin StructBiol 14:617-630), preferentially recognizes multiple Gal β1,4 GlcNAc(LacNAc) units which may be presented on the branches of N- or O-linkedglycans on cell surface glycoproteins such as CD45, CD43 and CD7(Stillman et al. (2006) J Immunol 176:778-789). Through binding andcrosslinking of specific glycoconjugates, Gal1 has the potential toinhibit T-cell effector functions and regulate the inflammatory response(Perillo et al. (1995) Nature 378:736-739; Rabinovich et al. (1999) JExp Med 190:385-397; Toscano et al. (2006) J Immunol 176:6323-6332;Santucci et al. (2003) Gastroenterol 124: 1381-1394; Baum et al. (2003)Clin Immunol 109:295-307). In several murine models of chronicinflammatory diseases, recombinant Gal1 suppressed Th1-dependentresponses and increased T-cell susceptibility to activation-induced celldeath (Rabinovich et al. (1999) J Exp Med 190:385-397; Toscano et al.(2006) J Immunol 176:6323-6332; Santucci et al. (2003) Gastroenterol124: 1381-1394; Baum et al. (2003) Clin Immunol 109:295-307).

In a recently described solid tumor (murine melanoma) model, Gal1 wasalso found to play a pivotal role in promoting escape fromT-cell-dependent immunity and conferring immune privilege to tumor cells(Rubinstein et al. (2004) Cancer Cell 5:241-251). In this model, Gal1blockade markedly enhanced syngeneic tumor rejection and tumor-specificT-cell-mediated immune responses (Rubinstein et al. (2004) Cancer Cell5:241-251). In another recently described solid tumor (head and necksquamous cell carcinomas), Gal1 overexpression was inversely correlatedwith the number of infiltrating T cells and was an independentprognostic factor for shorter overall survival (Le et al. (2005) J ClinOncol 23:8932-8941). WO2006/108474 describes the use of RNAi moleculesfor the treatment of cancer and non-Hodgkin's lymphoma.

In view of the above, it is clear that there remains a need in the artfor compositions and methods to combat immune disorders, includingHodgkin lymphoma. The present invention relates in general to a role ofGal1 in immune disorders, including Hodgkin lymphoma.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery thatgalectin-1 (Gal1) plays a role in immune disorders, including Hodgkinlymphoma. Accordingly, in one aspect, the invention provides for amethod for modulating an immune response by modulating the interactionbetween a Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s). In one embodiment, the method includes contacting an immunecell with an agent that modulates the interaction between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) tothereby modulate the immune response. In another embodiment, the immuneresponse is upregulated or downregulated. In yet another embodiment,signaling via the Gal1 binding partner is inhibited using an agent,e.g., a blocking antibody or an antigen binding fragment thereof thatrecognizes a Gal1 polypeptide or a fragment thereof and a blockingantibody, an antigen binding fragment thereof that recognizes the Gal1binding partner(s) or a fragment thereof, or an RNA interferencemolecule that downregulates Gal1. In still another embodiment, theimmune cell is contacted with an additional agent that upregulates animmune response. In other embodiments, the step of contacting occurs invivo or in vitro.

In another aspect, the invention provides for a method for treating asubject having a condition that would benefit from upregulation of animmune response. In one embodiment, the method includes administering anagent, e.g., a blocking antibody or an antigen binding fragment thereofthat recognizes a Gal1 polypeptide or a fragment thereof, a blockingantibody or an antigen binding fragment thereof that recognizes the Gal1binding partner(s) or a fragment thereof, or an RNA interferencemolecule that downregulates Gal1, that inhibits the interaction betweena Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment thereof on cells of a subject such that acondition that would benefit from upregulation of an immune response istreated. In another embodiment, the method further comprisesadministering a second agent that upregulates an immune response to thesubject. In yet another embodiment, the method further comprisesadministering a combination treatment, e.g., chemotherapy treatment. Ina further embodiment, the subject is a human. In yet a furtherembodiment, the human has Hodgkin lymphoma.

In another aspect, the invention features a method for detecting a Gal1polypeptide or nucleic acid or fragments thereof in a sample. In oneembodiment, the method includes contacting the sample with a compoundwhich selectively binds to a Gal1 polypeptide or fragment thereof anddetermining whether the compound binds to a Gal1 polypeptide or fragmentthereof in the sample to thereby detect the presence of a Gal1polypeptide or fragment thereof. In one embodiment, the compound whichbinds to the polypeptide is an antibody. In another aspect, the methodincludes contacting a sample with a nucleic acid probe or primer whichselectively hybridizes to a Gal1 polynucleotide or fragment thereof anddetermining whether the nucleic acid probe or primer binds to a nucleicacid molecule in the sample to thereby detect the presence of a Gal1polynucleotide or fragment thereof. In yet another embodiment, thesample comprises mRNA molecules and is contacted with a nucleic acidprobe.

In yet another aspect, the invention provides a method for identifying acompound which binds to a Gal1 polypeptide or fragment thereof. In onesuch embodiment, the method includes contacting a Gal1 polypeptide orfragment thereof, or a cell expressing said polypeptide with a testcompound and determining whether said polypeptide binds to the testcompound. In another embodiment, the binding of the test compound to aGal1 polypeptide or fragment thereof is detected by several methods,including detection of binding by direct detection of testcompound/polypeptide binding, detection of binding using a competitionbinding assay, and detection of binding using an assay for Gal1activity.

In still another aspect, the invention provides for a method formodulating the activity of a Gal1 polypeptide or fragment thereof. Inone embodiment, the method includes contacting the polypeptide or a cellexpressing the polypeptide with a compound which binds to thepolypeptide in a sufficient concentration to modulate the activity ofthe polypeptide.

In a further aspect, the invention provides for a method for identifyinga compound which modulates the activity of a Gal1 polypeptide orfragment thereof. In one embodiment, the method includes contacting aGal1 polypeptide or fragment thereof with a test compound anddetermining the effect of the test compound on the activity of thepolypeptide to thereby identify a compound which modulates the activityof the polypeptide.

In still another aspect, the invention provides for a cell-based assayfor screening for compounds which modulate the activity of Gal1. In oneembodiment, the assay includes contacting a cell expressing a Gal1binding partner(s) or fragment(s) thereof with a test compound anddetermining the ability of the test compound to modulate the activity ofthe Gal1 binding partner(s) or fragment(s) thereof. In anotherembodiment, the cell(s) are isolated from an animal model of an immunedisorder, e.g., a Hodgkin lymphoma animal model. In another embodiment,the cell(s) are isolated from a cell line associated with an immunedisorder, e.g., Hodgkin lymphoma cell line. In yet another embodiment,the cell(s) are isolated from a subject suffering from an immunedisorder, e.g., Hodgkin lymphoma.

In yet another aspect, the invention provides for a cell-free assay forscreening for compounds which modulate the binding of Gal1 or fragmentthereof to a Gal1 binding partner(s) or fragment(s) thereof. In oneembodiment, the assay includes contacting a Gal1 polypeptide or fragmentthereof with a test compound and determining the ability of the testcompound to bind to the Gal1 polypeptide or fragment thereof.

In one aspect, the invention provides for a method of assessing whethera subject has a condition, e.g., an immune disorder, including cancer,e.g., Hodgkin lymphoma, that would benefit from upregulation of animmune response. In one embodiment, the method includes comparing thelevel of expression of Gal1 in a subject sample and the normal level ofexpression of Gal1 in a control sample, wherein a significant increasein the level of expression of Gal1 in the subject sample relative to thenormal level is an indication that the subject is afflicted with acondition. In another embodiment, the sample comprises cells obtainedfrom the subject, for example, cells in fluid (e.g., whole blood fluid,serum fluid, plasma fluid, interstitial fluid, cerebrospinal fluid,lymph fluid, saliva, stool, and urine). In another embodiment, the levelof expression of Gal1 is assessed by detecting the presence in thesamples of a protein encoded by a Gal1 polynucleotide or a polypeptideor protein fragment thereof comprising the protein. For example, thepresence of the protein can be detected using a reagent whichspecifically binds to the protein, e.g., an antibody, an antibodyderivative, or an antibody fragment. In another embodiment, the level ofexpression of Gal1 is assessed by detecting the presence in the sampleof a transcribed polynucleotide encoded by a Gal1 polynucleotide or aportion of the transcribed polynucleotide, e.g., mRNA or cDNA. Forexample, the presence of the polynucleotide can be assayed by detectingthe presence in the sample of a transcribed polynucleotide which annealswith a Gal1 polynucleotide or anneals with a portion of a Gal1polynucleotide, under stringent hybridization conditions. In anotherembodiment, the transcribed polynucleotide to be detected can beamplified. In still another embodiment, a significant increase betweenthe level of expression of Gal1 in the subject sample relative to thenormal level of expression of Gal1 in the sample from the controlsubject can be at least about two, three, four, five, six, seven, eight,nine, ten, twenty or more fold greater.

In another aspect, the invention provides for a method for monitoringthe progression of an immune disorder, e.g., Hodgkin lymphoma, in asubject. In one embodiment, the method includes detecting in a subjectsample at a first point in time the expression of Gal1, repeating theprevious step at a subsequent point in time, and comparing the level ofexpression of Gal1 detected at each point in time to monitor theprogression of the immune disorder. In another embodiment, the subjectcan undergo treatment to ameliorate the immune disorder between thefirst point in time and the subsequent point in time. In one embodiment,the treatment may be chemotherapy. In yet another embodiment, thechemotherapy treatment may be combined with an agent.

In another aspect, the invention provides for a method for assessing theefficacy of a test compound for inhibiting an immune disorder, e.g.,Hodgkin lymphoma, in a subject. In one embodiment, the method includescomparing the level of expression of Gal1 in a first sample obtainedfrom the subject and exposed to the test compound and the level ofexpression of Gal1 in a second sample obtained from the subject, whereinthe second sample is not exposed to the test compound, and asignificantly lower level of expression of Gal1, relative to the secondsample, is an indication that the test compound is efficacious forinhibiting an immune disorder in the subject. In another embodiment, thefirst and second samples can be portions of a single sample obtainedfrom the subject or portions of pooled samples obtained from thesubject. In yet another embodiment, the method further comprisesadministering a combination treatment, wherein the treatment may includechemotherapy.

In another aspect, the invention provides for a method for predictingthe clinical outcome of a patient with an immune disorder, e.g., Hodgkinlymphoma. In one embodiment, the method includes determining the levelof expression of Gal1 in a patient sample, determining the level ofexpression of Gal1 in a sample from a control subject having a goodclinical outcome, and comparing the level of expression of Gal1 in thepatient sample and in the sample from the control subject, wherein asignificantly higher level of expression in the patient sample ascompared to the expression level in the sample from the control subjectis an indication that the patient has a poor clinical outcome.

In another aspect, the invention provides for a method of assessing theefficacy of a therapy for inhibiting an immune disorder, e.g., Hodgkinlymphoma, in a subject. In one embodiment, the method includes comparingthe level of expression of Gal1 in the first sample obtained from thesubject prior to providing at least a portion of the therapy to thesubject and the level of expression of Gal1 in a second sample obtainedfrom the subject following provision of the portion of the therapy,wherein a significantly lower level of expression of Gal1 in the secondsample, relative to the first sample, is an indication that the therapyis efficacious for inhibiting the immune disorder, e.g., Hodgkinlymphoma, in the subject.

In another aspect, the invention provides for methods of makingantibodies that specifically bind to a Gal1 polypeptide or a fragmentthereof. In one embodiment, the method involves making an isolatedhybridoma and includes immunizing a mammal using a compositioncomprising a Gal1 polypeptide or a fragment thereof, isolatingsplenocytes from the immunized mammal, fusing the isolated splenocyteswith an immortalized cell line to form hybridomas, and screeningindividual hybridomas for production of an antibody which specificallybinds with the polypeptide thereof to isolate the hybridoma. In anotherembodiment, the antibody or antigen binding fragment thereof produced bythe hybridoma can be used to specifically recognize Gal1 polypeptide ora fragment thereof. In still another embodiment, antibodies thatspecifically bind to a Gal1 polypeptide or a fragment thereof can bemade by immunizing a mammal with an effective amount of a preparation ofa material comprising a Gal1 polypeptide or a fragment thereof, incombination with an adjuvant.

In another aspect, the invention provides for novel compositions ofmatter that may be used in the methods of the invention. In oneembodiment, the invention provides antibodies or antigen bindingfragment thereof that specifically bind to a Gal1 polypeptide or afragment thereof. In one embodiment, the antibodies or antigen bindingfragment thereof can bind to a fragment of human Gal1, a polypeptidewhich is encoded by a nucleic acid comprising a nucleotide sequencewhich is at least 80% homologous to a nucleic acid comprising thenucleotide sequence human Gal1, or a polypeptide comprising an aminoacid sequence which is at least 80% homologous to the amino acidsequence of human Gal1. In other embodiments, the antibodies or antigenbinding portions thereof can be monoclonal, polyclonal, chimeric, orhumanized. In another embodiment, the antibodies or antigen bindingportions thereof can be detectably labeled. Non-limiting examples ofdetectable labels include an enzyme, a prosthetic group, a fluorescentmaterial, a luminescent material, a bioluminescent material, and aradioactive material. In other embodiments, the antibodies or antigenbinding portions thereof inhibit Hodgkin lymphoma in a subject. In yetanother embodiment, the antibodies or antigen binding portions thereofspecifically bind a Gal1 epitope comprising the ligand-specificcarbohydrate binding domain or fragment thereof, e.g., amino acids 30 to90 of human Gal1 or amino acids 62 to 86 of human Gal1. In anotherembodiment, the antibodies or antigen binding portion thereof cancomprise an effector domain and/or an Fc domain. In yet anotherembodiment, the antibodies or antigen binding portion thereof can besingle-chain antibodies and/or Fab fragments. In still anotherembodiment, a pharmaceutical composition comprising the antibodies orantigen binding portion thereof in a pharmaceutically acceptable carrierare provided.

In another aspect, the invention provides RNA interference compositionsand methods useful for the downregulation of Gal1 expression levels. Inone embodiment, an RNA interference molecule suitable for reducing theexpression of Gal1 comprises the sequence, GCTGCCAGATGGATACGAA (SEQ IDNO: 1), or a fragment or derivative thereof. In another embodiment, anexpression vector (e.g., an expression vector suitable for theproduction of double stranded RNA) comprises the sequence,GCTGCCAGATGGATACGAA (SEQ ID NO: 1), or a fragment or derivative thereofare provided. In another embodiment, the RNA interference compositionscan be used to treat an immune disorder, e.g., Hodgkin lymphoma.

In still another aspect, the invention provides for various kits, whichmay include the novel compositions described herein. In one embodiment,a kit is provided that comprises an agent which selectively binds to aGal1 polypeptide or fragment thereof and instructions for use. Inanother embodiment, a kit is provided that comprises an agent whichselectively hybridizes to a Gal1 polynucleotide or fragment thereof andinstructions for use. In yet another embodiment, the agent whichselectively hybridizes to a Gal1 polynucleotide or fragment thereof isan RNA interference molecule.

In yet another aspect, the invention provides for a vaccine comprisingan antigen and an agent that inhibits the interaction between Gal1 orfragment thereof and its natural binding partner(s) or fragment(s)thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D shows that Gal1 is overexpressed in classical Hodgkinlymphoma (cHL) cell lines and primary tumors. Relative Gal1 mRNAabundance (FIG. 1A and FIG. 1B) and protein expression (FIG. 1C) in apanel of LBCL and cHL cell lines is depicted. In FIG. 1A, the Gal1expression profiles of DLBCL, MLBCL and cHL cell lines are shown. Thecolor scale at the bottom of the figure indicates relative expressionand standard deviations from the mean. Red connotes high-levelexpression, while blue indicates low-level expression. In FIG. 1B, theplots represent the median expression of Gal1 (boxes) in LBCL versus cHLcell lines±25-75 percentile (bars) and ±range (whiskers). In FIG. 1C,the respective cHL cell lines (KMHZ, HDLM2, SupHD1, L1236, L540, L428,HD-MY-Z), the MLBCL cell line (Karpas 1106) and DLBCL cell lines (allothers) are indicated. FIG. 1D shows immunohistochemical (IHC) analysesof Gal1 in representative primary cHL (top panels) and DLBCL (bottompanels) cells (original magnification 40× and 400×, respectively).

FIGS. 2A-2E shows that Gal1 transcription is regulated by anAP1-dependent enhancer. FIG. 2A shows the results of analyses of theAP1-dependent Gal1 enhancer. The previously described Gal1 promoter(Salvatore et al. (1998) FEBS Lett 421:152-8) and putative enhancerelement including or lacking the predicted AP1 binding site (representedby a black bar) were cloned into a luciferase reporter vector,transiently transfected into cHL HD-MY-Z cells and assayed forluciferase activities. Representative luciferase activities from threeindependent experiments are normalized to Renilla luciferase activityand presented as bars±standard deviations. FIG. 2B shows results of theselective activity of the Gal1 enhancer. Classical HL, DLBCL andfibroblast cell lines were transfected with either the Gal1promoter-only vector (pGL3-Gal1⁻⁴⁰³⁺⁶⁷-Luc) or the promoter-enhancerconstruct (pGL3-Gal1₄₀₃₊₆₇-Luc-e₁₃₄₆₊₁₇₄₆) and assessed as in FIG. 2Afor their respective luciferase activities. FIG. 2C shows that the Gal1enhancer is dependent on AP-1 using electrophoretic mobility shiftassays. Nuclear extracts from DLBCL cell lines (DHL4, DHL7 and Toledo)or cHL cell lines (HD-MY-Z, L428 and SupHD1) were incubated with wildtype (WT) or mutant (MUT) ³²P labeled, double-stranded DNA probecorresponding to an AP1 binding site in the Gal1 enhancer. Specific,unlabeled competitor and antibodies against cJun or β-actin (control)were included in certain assays as indicated. The gel-shift bandcorresponding to probe-protein complex is indicated with an arrow andsupershift bands corresponding to probe-protein-antibody complex arenoted with asterisks. FIG. 2D shows that the Gal1 enhancer is dependenton cJUN. HD-MY-Z cells were cotransfected with the Gal1 promoter-onlyvector or the Gal1 promoter-enhancer construct with either thedominant-negative cJUN (cJUN-DN) construct (cJUN-DN) or empty vector.Luciferase activities were determined as in FIG. 2A. FIG. 2E shows thatinhibition of AP1 decreases Gal1 transcript abundance. HD-MY-Z cellswere transfected with either the dominant-negative cJUN construct(cJUN-DN) or empty vector and relative Gal1 mRNA abundance was thenassessed by RQ-PCR.

FIGS. 3A-3C shows that Gal1 confers immune privilege to cHLReed-Sternberg cells by favoring the expansion of Th2 cells and T_(reg)cells. FIG. 3A shows that Gal1 expression can be blocked in the cHLHD-MY-Z cell line using RNA interference (RNAi). HD-MY-Z cells weretransduced with pSIREN-RetroQ vector encoding Gal1-specific shRNA (Gal1shRNA, denoted as “G”) or scrambled control shRNA (SCR shRNA, denoted as“S”) and analyzed thereafter for Gal1 protein expression. FIG. 3B showsviability of total (CD3⁺) and CD4⁺ T cells co-cultured with Gal1 shRNAcHL or control SCR shRNA cHL cells. Following co-culture, T-cellviability was assessed using 3-color Annexin-V, -CD3 and -CD4 flowcytometry. FIG. 3C shows the relative abundance of the Th1- andTh2-specific transcription factors, Tbet and GATA3, in CD4⁺ cells fromthe Gal1 shRNA and SCR shRNA (control) cHL/T-cell co-cultures presentedin FIG. 3B. FIG. 3D shows the production of Th2 cytokines byGal1-treated T cells. Activated T cells were either untreated or treatedwith rGal1 in the presence or absence of TDG. Th2 cytokine (IL-4, IL-5,IL-10 and IL-13) production was then assessed using cytometric beadarrays. FIG. 3E shows the abundance of T_(reg) cells in Gal1-treated Tcells. Activated T cells were cultured in the presence of rGal1,rGal1+TDG or left untreated. The percentage of CD4⁺ CD25⁺ FOXP3⁺ T-cellswas then assessed by triple color-flow cytometry. Representativehistograms (left) and summary statistics (right) are shown.

FIG. 4 shows that recombinant Gal1 induces apoptosis in normal activatedT-cells. Activated T-cells were either untreated or treated with rGal1in the presence or absence of TDG. Thereafter, apoptosis was assessed byFITC-annexin V and PI double staining. The histograms (left panels) arerepresentative of 3 separate experiments that were averaged to obtainthe percent positive cells in the bar graphs (right panels).

FIG. 5 shows cJUN and JUN-B expression in LBCL and cHL cell lines. Therelative abundance of cJUN and JUN-B transcripts in DLBCL, MLBCL and cHLcell lines is shown. The color scale at the bottom indicates therelative expression and standard deviations from the mean. The plotsrepresent the median expression of Gal1 (horizontal line) in LBCL versuscHL cell lines±25-75 percentile (bars) and ±range (whiskers).Statistical differences in the relative cJUN and JUN-B expression inDLBCL and cHL cell lines were evaluated using a Mann-Whitney U test.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the discovery that galectin-1 (Gal1)is overexpressed by Reed-Sternberg (RS) cells associated with classicalHodgkin lymphomas (cHLs) and that the Gal1 overexpression by RS cells isdirectly implicated in the development and maintenance of animmunosuppressive Th2/T_(reg)-skewed microenvironment in cHL leading toan ineffective host anti-tumor immune response. Thus, agents such asnatural ligands, derivatives of natural ligands, and small molecules,RNA interference, aptamer, peptides, peptidomimetics, and antibodiesthat specifically bind to the Gal1 gene or gene products, or fragmentsthereof, can be utilized to modulate, e.g., increase, immunesurveillance in immune disorders, e.g., Hodgkin lymphoma. Additionally,agents such as Gal1 gene sequences, Gal1 gene products, anti-Gal1 RNAinterference molecules, anti-Gal1 antibodies (i.e., antibodies thatspecifically bind to Gal1 gene products or fragments thereof), orfragments thereof, can be utilized to reduce the level of TH2 cellactivity and/or increase the level of TH1 cell activity to restoreimmune surveillance in immune disorders, e.g., Hodgkin lymphoma.

Thus, it has been discovered that a higher than normal level ofexpression of Gal1 correlates with the presence of an immune disorder,e.g., Hodgkin lymphoma, in a patient. Gal1 polypeptides and fragmentsthereof, e.g., biologically active or antigenic fragments thereof, areprovided, as reagents or targets in assays applicable to treatmentand/or diagnosis of immune disorders, e.g., Hodgkin lymphoma. Inparticular, the methods and compositions of the present invention relateto detection and/or modulation of expression and/or activity of a Gal1gene or fragment thereof, e.g., biologically active fragments thereof,as well as to the detection and/or modulation of expression and/oractivity of gene products or fragments thereof encoded by the Gal1 gene,e.g., biologically active fragments thereof. The methods andcompositions of the present invention can utilize the Gal1 gene or genesequence or fragments thereof, as well as gene products of the Gal1 geneand/or modulators thereof or fragments thereof, e.g., antibodies whichspecifically bind to such Gal1 gene products.

In one aspect, methods are provided for detecting the presence, absence,stage, and other characteristics of immune disorders, e.g., Hodgkinlymphoma, in a sample that are relevant to prevention, diagnosis,characterization, and therapy in a patient.

The invention also features compositions of matter, including antibodies(e.g., antibodies which specifically bind to any one of the polypeptidesdescribed herein) as well as fusion polypeptides, including all or afragment of a polypeptide described herein. In addition, the inventionfeatures compositions useful for the reduction of Gal1 nucleic acids(e.g., Gal1 mRNA or hnRNA or fragments thereof), including RNAinterference compositions, directed against Gal1 nucleic acids orfragments thereof.

I. Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

Unless otherwise specified here within, the terms “antibody” and“antibodies” broadly encompass naturally-occurring forms of antibodies(e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such assingle-chain antibodies, chimeric and humanized antibodies andmulti-specific antibodies, as well as fragments and derivatives of allof the foregoing, which fragments and derivatives have at least anantigenic binding site. Antibody derivatives may comprise a protein orchemical moiety conjugated to an antibody.

The term “antibody” as used herein also includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen (e.g., Gal1 polypeptide or fragment thereof). It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. Examples of binding fragmentsencompassed within the term “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) aFv fragment consisting of the VL and VH domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a VH domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent polypeptides (known as single chain Fv (scFv);see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al.(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998,Nature Biotechnology 16: 778). Such single chain antibodies are alsointended to be encompassed within the term “antigen-binding portion” ofan antibody. Any VH and VL sequences of specific scFv can be linked tohuman immunoglobulin constant region cDNA or genomic sequences, in orderto generate expression vectors encoding complete IgG polypeptides orother isotypes. VH and VL can also be used in the generation of Fab, Fvor other fragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of larger immunoadhesion polypeptides, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionpolypeptides include use of the streptavidin core region to make atetrameric scFv polypeptide (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv polypeptides (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionpolypeptides can be obtained using standard recombinant DNA techniques,as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, orsyngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.).Antibodies may also be fully human. Preferably, antibodies of theinvention bind specifically or substantially specifically to Gal1polypeptides or fragments thereof. The terms “monoclonal antibodies” and“monoclonal antibody composition”, as used herein, refer to a populationof antibody polypeptides that contain only one species of an antigenbinding site capable of immunoreacting with a particular epitope of anantigen, whereas the term “polyclonal antibodies” and “polyclonalantibody composition” refer to a population of antibody polypeptidesthat contain multiple species of antigen binding sites capable ofinteracting with a particular antigen. A monoclonal antibody compositiontypically displays a single binding affinity for a particular antigenwith which it immunoreacts.

The term “body fluid” refers to fluids that are excreted or secretedfrom the body as well as fluid that are normally not (e.g. amnioticfluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid,cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle,chyme, stool, female ejaculate, interstitial fluid, intracellular fluid,lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum,semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication,vitreous humor, vomit).

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

A molecule is “fixed” or “affixed” to a substrate if it is covalently ornon-covalently associated with the substrate such the substrate can berinsed with a fluid (e.g. standard saline citrate, pH 7.4) without asubstantial fraction of the molecule dissociating from the substrate.

“Homologous” as used herein, refers to nucleotide sequence similaritybetween two regions of the same nucleic acid strand or between regionsof two different nucleic acid strands. When a nucleotide residueposition in both regions is occupied by the same nucleotide residue,then the regions are homologous at that position. A first region ishomologous to a second region if at least one nucleotide residueposition of each region is occupied by the same residue. Homologybetween two regions is expressed in terms of the proportion ofnucleotide residue positions of the two regions that are occupied by thesame nucleotide residue. By way of example, a region having thenucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotidesequence 5′-TATGGC-3′ share 50% homology. Preferably, the first regioncomprises a first portion and the second region comprises a secondportion, whereby, at least about 50%, and preferably at least about 75%,at least about 90%, or at least about 95% of the nucleotide residuepositions of each of the portions are occupied by the same nucleotideresidue. More preferably, all nucleotide residue positions of each ofthe portions are occupied by the same nucleotide residue.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid of the invention, such as a recombinant expressionvector of the invention, has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It should beunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The term “humanized antibody”, as used herein, is intended to includeantibodies made by a non-human cell having variable and constant regionswhich have been altered to more closely resemble antibodies that wouldbe made by a human cell. For example, by altering the non-human antibodyamino acid sequence to incorporate amino acids found in human germlineimmunoglobulin sequences. The humanized antibodies of the invention mayinclude amino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs. The term “humanized antibody”, as used herein, also includesantibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto humanframework sequences.

As used herein, the term “immune cell” refers to cells that play a rolein the immune response. Immune cells are of hematopoietic origin, andinclude lymphocytes, such as B cells and T cells; natural killer cells;myeloid cells, such as monocytes, macrophages, eosinophils, mast cells,basophils, and granulocytes.

As used herein, the term “immune disorder” includes immune diseases,conditions, and predispositions to, including, but not limited to,Hodgkin lymphoma (including, e.g., lymphocyte-rich classical Hodgkinlymphoma, mixed cellularity classical Hodgkin lymphoma,lymphocyte-depleted classical Hodgkin lymphoma, nodular sclerosisclassical Hodgkin lymphoma, and nodular lymphocyte predominant Hodgkinlymphoma), cancer, chronic inflammatory disease and disorders(including, e.g., Crohn's disease, inflammatory bowel disease, reactivearthritis, and Lyme disease), insulin-dependent diabetes, organ specificautoimmunity (including, e.g., multiple sclerosis, Hashimoto'sthyroiditis, autoimmune uveitis, and Grave's disease), contactdermatitis, psoriasis, graft rejection, graft versus host disease,sarcoidosis, atopic conditions (including, e.g., asthma and allergyincluding, but not limited to, allergic rhinitis and gastrointestinalallergies such as food allergies), eosinophilia, conjunctivitis,glomerular nephritis, systemic lupus erythematosus, scleroderma, certainpathogen susceptibilities such as helminthic (including, e.g.,leishmaniasis) and certain viral infections (including, e.g., HIV andbacterial infections such as tuberculosis and lepromatous leprosy).

As used herein, the term “immune response” includes T cell mediatedand/or B cell mediated immune responses. Exemplary immune responsesinclude T cell responses, e.g., cytokine production, and cellularcytotoxicity. In addition, the term immune response includes immuneresponses that are indirectly effected by T cell activation, e.g.,antibody production (humoral responses) and activation of cytokineresponsive cells, e.g., macrophages.

As used herein, the term “inhibit” includes the decrease, limitation, orblockage, of, for example a particular action, function, or interaction.

As used herein, the term “interaction”, when referring to an interactionbetween two molecules, refers to the physical contact (e.g., binding) ofthe molecules with one another. Generally, such an interaction resultsin an activity (which produces a biological effect) of one or both ofsaid molecules. The activity may be a direct activity of one or both ofthe molecules, (e.g., signal transduction). Alternatively, one or bothmolecules in the interaction may be prevented from binding their ligand,and thus be held inactive with respect to ligand binding activity (e.g.,binding its ligand and triggering or inhibiting an immune response). Toinhibit such an interaction results in the disruption of the activity ofone or more molecules involved in the interaction. To enhance such aninteraction is to prolong or increase the likelihood of said physicalcontact, and prolong or increase the likelihood of said activity.

As used herein, an “antisense” nucleic acid polypeptide comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA polypeptide, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid polypeptide can hydrogen bond to a sense nucleic acidpolypeptide.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds Gal1 polypeptide or a fragment thereof is substantially free ofantibodies that specifically bind antigens other than a Gal1 polypeptideor a fragment thereof). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material, separationmedium, and culture medium when isolated from cells or produced byrecombinant DNA techniques, or chemical precursors or other chemicalswhen chemically synthesized. An “isolated” or “purified” protein orbiologically active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue sourcefrom which the antibody, polypeptide, peptide or fusion protein isderived, or substantially free from chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of Gal1 polypeptide orfragment thereof, in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of Gal1 protein or fragmentthereof, having less than about 30% (by dry weight) of non-Gal1 protein(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-Gal1 protein, still more preferably less thanabout 10% of non-Gal1 protein, and most preferably less than about 5%non-Gal1 protein. When antibody, polypeptide, peptide or fusion proteinor fragment thereof, e.g., a biologically active fragment thereof, isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

A “kit” is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g. a probe, for specifically detecting theexpression of a marker of the invention. The kit may be promoted,distributed, or sold as a unit for performing the methods of the presentinvention.

A “marker” is a gene whose altered level of expression in a tissue orcell from its expression level in normal or healthy tissue or cell isassociated with a disease state, such as cancer. A “marker nucleic acid”is a nucleic acid (e.g., mRNA, cDNA) encoded by or corresponding to amarker of the invention. Such marker nucleic acids include DNA (e.g.,cDNA) comprising the entire or a partial sequence of any of the nucleicacid sequences set forth in the Sequence Listing or the complement ofsuch a sequence. The marker nucleic acids also include RNA comprisingthe entire or a partial sequence of any of the nucleic acid sequencesset forth in the Sequence Listing or the complement of such a sequence,wherein all thymidine residues are replaced with uridine residues. A“marker protein” is a protein encoded by or corresponding to a marker ofthe invention. A marker protein comprises the entire or a partialsequence of any of the sequences set forth in the Sequence Listing. Theterms “protein” and “polypeptide” are used interchangeably.

As used herein, the term “modulate” includes up-regulation anddown-regulation, e.g., enhancing or inhibiting a response.

The “normal” level of expression of a marker is the level of expressionof the marker in cells of a subject, e.g., a human patient, notafflicted with an immune disorder, e.g., Hodgkin lymphoma. An“over-expression” or “significantly higher level of expression” of amarker refers to an expression level in a test sample that is greaterthan the standard error of the assay employed to assess expression, andis preferably at least twice, and more preferably three, four, five orten times the expression level of the marker in a control sample (e.g.,sample from a healthy subjects not having the marker associated disease)and preferably, the average expression level of the marker in severalcontrol samples. A “significantly lower level of expression” of a markerrefers to an expression level in a test sample that is at least twice,and more preferably three, four, five or ten times lower than theexpression level of the marker in a control sample (e.g., sample from ahealthy subject not having the marker associated disease) andpreferably, the average expression level of the marker in severalcontrol samples.

The term “probe” refers to any molecule which is capable of selectivelybinding to a specifically intended target molecule, for example, anucleotide transcript or protein encoded by or corresponding to amarker. Probes can be either synthesized by one skilled in the art, orderived from appropriate biological preparations. For purposes ofdetection of the target molecule, probes may be specifically designed tobe labeled, as described herein. Examples of molecules that can beutilized as probes include, but are not limited to, RNA, DNA, proteins,antibodies, and organic molecules.

As used herein, “subject” refers to any healthy animal, mammal or human,or any animal, mammal or human afflicted with an immune disorder, e.g.,Hodgkin lymphoma. The term “subject” is interchangeable with “patient”.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of antibody, polypeptide, peptide orfusion protein in which the protein is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. In one embodiment, the language “substantially free of chemicalprecursors or other chemicals” includes preparations of antibody,polypeptide, peptide or fusion protein having less than about 30% (bydry weight) of chemical precursors or non-antibody, polypeptide, peptideor fusion protein chemicals, more preferably less than about 20%chemical precursors or non-antibody, polypeptide, peptide or fusionprotein chemicals, still more preferably less than about 10% chemicalprecursors or non-antibody, polypeptide, peptide or fusion proteinchemicals, and most preferably less than about 5% chemical precursors ornon-antibody, polypeptide, peptide or fusion protein chemicals.

A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or homologous with all or a portion of amature mRNA made by transcription of a marker of the invention andnormal post-transcriptional processing (e.g. splicing), if any, of theRNA transcript, and reverse transcription of the RNA transcript.

As used herein, the term “T cell” includes CD4+ T cells and CD8+ Tcells. The term T cell also includes both T helper 1 type T cells and Thelper 2 type T cells. The term “antigen presenting cell” includesprofessional antigen presenting cells (e.g., B lymphocytes, monocytes,dendritic cells, Langerhans cells) as well as other antigen presentingcells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts,oligodendrocytes).

As used herein, the term “vector” refers to a nucleic acid capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” or simply “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid and the amino acidsequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA,ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAGGlutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGTHistidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine(Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signal(end) TAA, TAG, TGA

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNA codingfor a fusion protein or polypeptide of the invention (or any portionthereof) can be used to derive the fusion protein or polypeptide aminoacid sequence, using the genetic code to translate the DNA or RNA intoan amino acid sequence. Likewise, for fusion protein or polypeptideamino acid sequence, corresponding nucleotide sequences that can encodethe fusion protein or polypeptide can be deduced from the genetic code(which, because of its redundancy, will produce multiple nucleic acidsequences for any given amino acid sequence). Thus, description and/ordisclosure herein of a nucleotide sequence which encodes a fusionprotein or polypeptide should be considered to also include descriptionand/or disclosure of the amino acid sequence encoded by the nucleotidesequence. Similarly, description and/or disclosure of a fusion proteinor polypeptide amino acid sequence herein should be considered to alsoinclude description and/or disclosure of all possible nucleotidesequences that can encode the amino acid sequence.

I. Description

The present invention relates to methods and compositions for thetreatment and diagnosis of immune disorders, especially Tlymphocyte-related disorders, including, but not limited to, Hodgkinlymphoma (including, e.g., lymphocyte-rich classical Hodgkin lymphoma,mixed cellularity classical Hodgkin lymphoma, lymphocyte-depletedclassical Hodgkin lymphoma, nodular sclerosis classical Hodgkinlymphoma, and nodular lymphocyte predominant Hodgkin lymphoma), cancer,chronic inflammatory disease and disorders (including, e.g., Crohn'sdisease, inflammatory bowel disease, reactive arthritis, and Lymedisease), insulin-dependent diabetes, organ specific autoimmunity(including, e.g., multiple sclerosis, Hashimoto's thyroiditis,autoimmune uveitis, and Grave's disease), contact dermatitis, psoriasis,graft rejection, graft versus host disease, sarcoidosis, atopicconditions (including, e.g., asthma and allergy including, but notlimited to, allergic rhinitis and gastrointestinal allergies such asfood allergies), eosinophilia, conjunctivitis, glomerular nephritis,systemic lupus erythematosus, scleroderma, certain pathogensusceptibilities such as helminthic (including, e.g., leishmaniasis) andcertain viral infections (including, e.g., HIV and bacterial infectionssuch as tuberculosis and lepromatous leprosy).

In particular, the methods and compositions of the present inventionrelate to detection and/or modulation of expression and/or activity of agene referred to herein as the galectin-1 (Gal1) gene or a fragmentthereof, e.g., a biologically active fragment thereof, as well as to thedetection and/or modulation of expression and/or activity of geneproducts encoded by the Gal1 gene (i.e., a “Gal1 gene product”) orfragments thereof, e.g., biologically active fragments thereof. Themethods and compositions of the present invention can utilize the Gal1gene or gene sequence or fragments thereof, as well as gene products ofthe Gal1 gene and/or modulators thereof, e.g., antibodies whichspecifically bind to such Gal1 gene products, or fragments thereof.Sequences, structures, domains, biophysical characteristics, andfunctions of Gal1 gene and gene products have been described in the art.See, for example, Rabinovich et al. (2002) Trends Immunol 23:313-320;Liu and Rabinovich (2005) Nature Reviews Cancer 5:29-41; Rubinstein etal. (2004) Cancer Cell 5:241-251; Le et al. (2005) J Clin Oncol23:8932-8941; Vasta et al. (2004) Curr Opin Struct Biol 14:617-630;Toscano et al. (2007) Cyt Growth Fact Rev 18:57-71; Camby et al. (2006)Glycobiol 16:137R-157R, each of which is incorporated herein, byreference, in its entirety. Gal1 gene and gene products from manyspecies are known and include, for example, chimpanzee Gal1 (NCBIAccession XM_(—)001162066), rat Gal1 (NCBI Accession NM_(—)019904),mouse Gal1 (NM_(—)008495), and chicken Gal1 (NM_(—)205495). Human Gal1sequences include those listed below.

Gal1 coding nucleic acid sequence: (SEQ ID NO: 2)ATGGCTTGTG GTCTGGTCGC CAGCAACCTG AATCTCAAACCTGGAGAGTG CCTTCGAGTG CGAGGCGAGG TGGCTCCTGACGCTAAGAGC TTCGTGCTGA ACCTGGGCAA AGACAGCAACAACCTGTGCC TGCACTTCAA CCCTCGCTTC AACGCCCACGGCGACGCCAA CACCATCGTG TGCAACAGCA AGGACGGCGGGGCCTGGGGG ACCGAGCAGC GGGAGGCTGT CTTTCCCTTCCAGCCTGGAA GTGTTGCAGA GGTGTGCATC ACCTTCGACCAGGCCAACCT GACCGTCAAG CTGCCAGATG GATACGAATTCAAGTTCCCC AACCGCCTCA ACCTGGAGGC CATCAACTACATGGCAGCTG ACGGTGACTT CAAGATCAAA TGTGTGGCCT TTGACTGA  Gal1 protein sequence: (SEQ ID NO: 3)MACGLVASNL NLKPGECLRV RGEVAPDAKS FVLNLGKDSNNLCLHFNPRF NAHGDANTIV CNSKDGGAWG TEQREAVFPFQPGSVAEVCI TFDQANLTVK LPDGYEFKFP NRLNLEAINY MAADGDFKIK CVAFD 

The invention is based, in part, on the discovery that Gal1 isoverexpressed by Reed-Sternberg (RS) cells associated with classicalHodgkin lymphomas (cHLs) and that the Gal1 overexpression by RS cells isdirectly implicated in the development and maintenance of animmunosuppressive Th2/T_(reg)-skewed microenvironment in cHL leading toan ineffective host anti-tumor immune response. Thus, agents such asnatural ligands, derivatives of natural ligands, and small molecules,RNA interference, aptamer, peptides, peptidomimetics, and antibodiesthat specifically bind to the Gal1 gene or gene products or fragmentsthereof can be utilized to modulate (e.g., increase) immune surveillancein immune disorders, e.g., Hodgkin lymphoma. Additionally, agents suchas Gal1 gene sequences, Gal1 gene products, anti-Gal1 RNA interferencemolecules, anti-Gal1 antibodies (i.e., antibodies that specifically bindto Gal1 gene products), or fragments thereof, can be utilized to reducethe level of TH2 cell activity and/or increase the level of TH1 cellactivity to restore immune surveillance in immune disorders, e.g.,Hodgkin lymphoma.

The Gal1 gene is also expressed in other cells known in the art. See,for example, Rabinovich et al. (2002) Trends Immunol 23:313-320; Liu andRabinovich (2005) Nature Reviews Cancer 5:29-41; Rubinstein et al.(2004) Cancer Cell 5:241-251; Le et al. (2005) J Clin Oncol23:8932-8941; Vasta et al. (2004) Curr Opin Struct Biol 14:617-630;Toscano et al. (2007) Cyt Growth Fact Rev 18:57-71; Camby et al. (2006)Glycobiol 16:137R-157R, each of which is incorporated herein, byreference, in its entirety. Thus, the above-described compositions(e.g., natural ligands, derivatives of natural ligands, and smallmolecules, RNA interference, aptamer, peptides, peptidomimetics,antibodies that specifically bind to the Gal1 gene or gene products, orfragments thereof) can also be utilized to modulate immune responses inthese immune related cell. Additional studies indicate that Gal1 is alsooverexpressed in other hematologic malignancies, including certainsubtypes of childhood acute lymphoblastic leukemia with adverseprognosis, and can be utilized as a diagnostic and prognostic marker inthese diseases (Armstrong et al. (2002) Nat Genet. 30:41-47).

II. Agents that Modulate Immune Cell Activation

The agents of this invention can modulate, e.g., up or down regulate,expression and/or activity of gene products or fragments thereof encodedby the Gal1 gene or fragment thereof and, thereby, modulate, e.g., up ordownregulate, an immune response. For example, overexpression of Gal1 bycHL RS cells decreases the viability of activated T cells and skews thebalance towards a Th2 immune response (e.g., by increasing the secretionof Th2 cytokines including IL-4, IL-5, IL-10 and IL-13) and fosters theexpansion and/or retention of CD4⁺ CD25^(high) FOXP3⁺ T_(reg) cells. Theinteraction between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof in the context ofcHL results in an immunosuppressive Th2/T_(reg)-skewed microenvironment.Thus, in one embodiment, agents which block the interactions between aGal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof can enhance an immune response(e.g., restore immune surveillance in cHL). In another embodiment,agents that increase the interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof can decrease an immune response (e.g., immunosuppression).Exemplary agents for modulating a Gal1-mediated immune response includeantibodies against Gal1 which inhibit the interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof, small molecules, peptides, peptidomimetics,natural ligands, and derivatives of natural ligands, which inhibit theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof, and RNAinterference, antisense, and nucleic acid aptamers that reduce Gal1nucleic acids or Gal1 expression products or fragments thereof.

An isolated Gal1 polypeptide or a fragment thereof (or a nucleic acidencoding such a polypeptide), can be used as an immunogen to generateantibodies that bind to said immunogen, using standard techniques forpolyclonal and monoclonal antibody preparation. A full-length Gal1polypeptide can be used, or alternatively, the invention relates toantigenic peptide fragments of Gal1 polypeptide for use as immunogens.An antigenic peptide of Gal1 comprises at least 8 amino acid residuesand encompasses an epitope present in the respective full lengthmolecule such that an antibody raised against the peptide forms aspecific immune complex with the respective full length molecule.Preferably, the antigenic peptide comprises at least 10 amino acidresidues. Preferred epitopes encompassed by the antigenic peptides areregions of Gal1 that mediate ligand specific carbohydrate binding, e.g.,the Gal1 carbohydrate recognition domain, amino acids 30 to 90 of humanGal1, and amino acids 62 to 86 of human Gal1. In one embodiment suchepitopes can be specific for a given polypeptide molecule from onespecies, such as mouse or human (i.e., an antigenic peptide that spans aregion of the polypeptide molecule that is not conserved across speciesis used as immunogen; such non conserved residues can be determinedusing an alignment such as that provided herein).

In one embodiment, an antibody binds substantially specifically to aGal1 polypeptide, or a fragment thereof. In a preferred embodiment, anantibody binds to a Gal1 polypeptide, or a fragment thereof, and blocksthe interaction between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof.

A Gal1 immunogen typically is used to prepare antibodies by immunizing asuitable subject (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, a recombinantly expressed or chemically synthesized molecule orfragment thereof to which the immune response is to be generated. Thepreparation can further include an adjuvant, such as Freund's completeor incomplete adjuvant, or similar immunostimulatory agent. Immunizationof a suitable subject with an immunogenic preparation induces apolyclonal antibody response to the antigenic peptide contained therein.

Polyclonal antibodies can be prepared as described above by immunizing asuitable subject with a polypeptide immunogen. The polypeptide antibodytiter in the immunized subject can be monitored over time by standardtechniques, such as with an enzyme linked immunosorbent assay (ELISA)using immobilized polypeptide. If desired, the antibody directed againstthe antigen can be isolated from the mammal (e.g., from the blood) andfurther purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also Brown et al. (1981) J. Immunol.127:539-46; Brown et al (1980) J. Biol. Chem. 255:4980-83; Yeh et al(1976) Proc. Natl. Acad. Sci. 76:2927-31; and Yeh et al. (1982) Int. J.Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique(Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generallyKenneth, R. H. in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); Lerner, E. A.(1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with an immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds to the polypeptideantigen, preferably specifically.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-Gal1 monoclonal antibody (see, e.g., Galfre, G. et al. (1977)Nature 266:55052; Gefter et al. (1977) supra; Lerner (1981) supra;Kenneth (1980) supra). Moreover, the ordinary skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag-4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from theAmerican Type Culture Collection (ATCC), Rockville, Md. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bind agiven polypeptide, e.g., using a standard ELISA assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas,a monoclonal specific for one of the above described polypeptidesantibody can be identified and isolated by screening a recombinantcombinatorial immunoglobulin library (e.g., an antibody phage displaylibrary) with the appropriate polypeptide to thereby isolateimmunoglobulin library members that bind the polypeptide. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening an antibodydisplay library can be found in, for example, Ladner et al. U.S. Pat.No. 5,223,409; Kang et al. International Publication No. WO 92/18619;Dower et al International Publication No. WO 91/17271; Winter et alInternational Publication WO 92/20791; Markland et al. InternationalPublication No. WO 92/15679; Breitling et al. International PublicationWO 93/01288; McCafferty et al International Publication No. WO 92/01047;Garrard et al. International Publication No. WO 92/09690; Ladner et alInternational Publication No. WO 90/02809; Fuchs et al. (1991)Biotechnology (NY) 9:1369-1372; Hay et al (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clarkson et al (1991) Nature 352:624-628; Gram et al (1992)Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991)Biotechnology (NY) 9:1373-1377; Hoogenboom et al. (1991) Nucleic AcidsRes. 19:4133-4137; Barbas et al (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al (1990) Nature 348:552-554.

Additionally, recombinant anti-Gal1 polypeptide antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al. International Patent Publication PCT/US86/02269; Akiraet al. European Patent Application 184,187; Taniguchi, M. EuropeanPatent Application 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT Application WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218;Nishimura et al (1987) Cancer Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) Biotechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

In addition, humanized antibodies can be made according to standardprotocols such as those disclosed in U.S. Pat. No. 5,565,332. In anotherembodiment, antibody chains or specific binding pair members can beproduced by recombination between vectors comprising nucleic acidmolecules encoding a fusion of a polypeptide chain of a specific bindingpair member and a component of a replicable generic display package andvectors containing nucleic acid molecules encoding a second polypeptidechain of a single binding pair member using techniques known in the art,e.g., as described in U.S. Pat. No. 5,565,332, 5,871,907, or 5,733,743.The use of intracellular antibodies to inhibit protein function in acell is also known in the art (see e.g., Carlson, J. R. (1988) Mol.Cell. Biol. 8:2638-2646; Biocca, S. et al (1990) EMBO J. 9:101-108;Werge, T. M. et al. (1990) FEBS Lett. 274:193-198; Carlson, J. R. (1993)Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W. A. et al. (1993)Proc. Natl. Acad. Sci. USA 90:7889-7893; Biocca, S. et al. (1994)Biotechnology (NY) 12:396-399; Chen, S-Y. et al. (1994) Hum. Gene Ther.5:595-601; Duan, L et al. (1994) Proc. Natl. Acad. Sci. USA91:5075-5079; Chen, S-Y. et al (1994) Proc. Natl. Acad. Sci. USA91:5932-5936; Beerli, R. R. et al. (1994) J. Biol. Chem.269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys. Res.Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al).

Additionally, fully human antibodies could be made against a Gal1immunogen. Fully human antibodies can be made in mice that aretransgenic for human immunoglobulin genes, e.g. according to Hogan, etal., “Manipulating the Mouse Embryo: A Laboratory Manuel,” Cold SpringHarbor Laboratory. Briefly, transgenic mice are immunized with purifiedGal1 immunogen. Spleen cells are harvested and fused to myeloma cells toproduce hybridomas. Hybridomas are selected based on their ability toproduce antibodies which bind to the Gal1 immunogen. Fully humanantibodies would reduce the immunogenicity of such antibodies in ahuman.

In one embodiment, an antibody for use in the instant invention is abispecific antibody. A bispecific antibody has binding sites for twodifferent antigens within a single antibody polypeptide. Antigen bindingmay be simultaneous or sequential. Triomas and hybrid hybridomas are twoexamples of cell lines that can secrete bispecific antibodies. Examplesof bispecific antibodies produced by a hybrid hybridoma or a trioma aredisclosed in U.S. Pat. No. 4,474,893. Bispecific antibodies have beenconstructed by chemical means (Staerz et al. (1985) Nature 314:628, andPerez et al. (1985) Nature 316:354) and hybridoma technology (Staerz andBevan (1986) Proc. Natl. Acad. Sci. USA, 83:1453, and Staerz and Bevan(1986) Immunol. Today 7:241). Bispecific antibodies are also describedin U.S. Pat. No. 5,959,084. Fragments of bispecific antibodies aredescribed in U.S. Pat. No. 5,798,229.

Bispecific agents can also be generated by making heterohybridomas byfusing hybridomas or other cells making different antibodies, followedby identification of clones producing and co-assembling both antibodies.They can also be generated by chemical or genetic conjugation ofcomplete immunoglobulin chains or portions thereof such as Fab and Fvsequences. The antibody component can bind to a Gal1 polypeptide or afragment thereof. In one embodiment, the bispecific antibody couldspecifically bind to both a Gal1 polypeptide or a fragment thereof andits natural binding partner(s) or a fragment(s) thereof.

Yet another aspect of the invention pertains to anti-Gal1 antibodiesthat are obtainable by a process comprising, immunizing an animal withan immunogenic Gal1 polypeptide or an immunogenic portion thereof uniqueto Gal1; and then isolating from the animal antibodies that specificallybind to the polypeptide or a fragment thereof.

In another aspect of this invention, peptides or peptide mimetics can beused to antagonize or promote the interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof. In one embodiment, variants of Gal1 whichfunction as a modulating agent for the respective full length protein,can be identified by screening combinatorial libraries of mutants, e.g.,truncation mutants, for antagonist activity. In one embodiment, avariegated library of Gal1 variants is generated by combinatorialmutagenesis at the nucleic acid level and is encoded by a variegatedgene library. A variegated library of Gal1 variants can be produced, forinstance, by enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential polypeptide sequences is expressible as individualpolypeptides containing the set of polypeptide sequences therein. Thereare a variety of methods which can be used to produce libraries ofpolypeptide variants from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene then ligated into anappropriate expression vector. Use of a degenerate set of genes allowsfor the provision, in one mixture, of all of the sequences encoding thedesired set of potential polypeptide sequences. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem.53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983)Nucleic Acid Res. 11:477.

In addition, libraries of fragments of a polypeptide coding sequence canbe used to generate a variegated population of polypeptide fragments forscreening and subsequent selection of variants of a given polypeptide.In one embodiment, a library of coding sequence fragments can begenerated by treating a double stranded PCR fragment of a polypeptidecoding sequence with a nuclease under conditions wherein nicking occursonly about once per polypeptide, denaturing the double stranded DNA,renaturing the DNA to form double stranded DNA which can includesense/antisense pairs from different nicked products, removing singlestranded portions from reformed duplexes by treatment with S1 nuclease,and ligating the resulting fragment library into an expression vector.By this method, an expression library can be derived which encodesN-terminal, C-terminal and internal fragments of various sizes of thepolypeptide.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of polypeptides. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofGal1 (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;Delagrave et al. (1993) Protein Eng. 6(3):327-331). In one embodiment,cell based assays can be exploited to analyze a variegated polypeptidelibrary. For example, a library of expression vectors can be transfectedinto a cell line which ordinarily synthesizes Gal1. The transfectedcells are then cultured such that the full length polypeptide and aparticular mutant polypeptide are produced and the effect of expressionof the mutant on the full length polypeptide activity in cellsupernatants can be detected, e.g., by any of a number of functionalassays. Plasmid DNA can then be recovered from the cells which score forinhibition, or alternatively, potentiation of full length polypeptideactivity, and the individual clones further characterized.

Systematic substitution of one or more amino acids of a polypeptideamino acid sequence with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) can be used to generate more stable peptides. Inaddition, constrained peptides comprising a polypeptide amino acidsequence of interest or a substantially identical sequence variation canbe generated by methods known in the art (Rizo and Gierasch (1992) Annu.Rev. Biochem. 61:387, incorporated herein by reference); for example, byadding internal cysteine residues capable of forming intramoleculardisulfide bridges which cyclize the peptide.

The amino acid sequences disclosed herein will enable those of skill inthe art to produce polypeptides corresponding peptide sequences andsequence variants thereof. Such polypeptides can be produced inprokaryotic or eukaryotic host cells by expression of polynucleotidesencoding the peptide sequence, frequently as part of a largerpolypeptide. Alternatively, such peptides can be synthesized by chemicalmethods. Methods for expression of heterologous proteins in recombinanthosts, chemical synthesis of polypeptides, and in vitro translation arewell known in the art and are described further in Maniatis et al.Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold SpringHarbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152,Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; ChaikenI. M. (1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989)Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H.(1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980)Semisynthetic Proteins, Wiley Publishing, which are incorporated hereinby reference).

In one embodiment, the peptide has an amino acid sequence identical orsimilar to the Gal1 binding site of its natural binding partner(s) or afragment(s) thereof. In one embodiment, the peptide competes with a Gal1polypeptide or a fragment thereof for binding its natural bindingpartner(s) or a fragment(s) thereof. In a preferred embodiment, thepeptide carries carbohydrate moieties recognized by a Gal1 polypeptideor a fragment thereof and said peptide competes with the Gal1polypeptide or a fragment thereof for binding the Gal1 natural bindingpartner(s) or a fragment(s) thereof.

Peptides can be produced, typically by direct chemical synthesis, andused e.g., as antagonists of the interactions between a Gal1 polypeptideor a fragment thereof and its natural binding partner(s) or afragment(s) thereof. Peptides can be produced as modified peptides, withnonpeptide moieties attached by covalent linkage to the N-terminusand/or C-terminus. In certain preferred embodiments, either thecarboxy-terminus or the amino-terminus, or both, are chemicallymodified. The most common modifications of the terminal amino andcarboxyl groups are acetylation and amidation, respectively.Amino-terminal modifications such as acylation (e.g., acetylation) oralkylation (e.g., methylation) and carboxy-terminal-modifications suchas amidation, as well as other terminal modifications, includingcyclization, can be incorporated into various embodiments of theinvention. Certain amino-terminal and/or carboxy-terminal modificationsand/or peptide extensions to the core sequence can provide advantageousphysical, chemical, biochemical, and pharmacological properties, suchas: enhanced stability, increased potency and/or efficacy, resistance toserum proteases, desirable pharmacokinetic properties, and others.Peptides disclosed herein can be used therapeutically to treat disease,e.g., by altering costimulation in a patient.

Peptidomimetics (Fauchere, J. (1986) Adv. Drug Res. 15:29; Veber andFreidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem.30:1229, which are incorporated herein by reference) are usuallydeveloped with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides can be used to produce an equivalent therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as a human Gal1 polypeptide or afragment thereof, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: —CH2NH—,—CH2S—, —CH2-CH2-, —CH═CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and—CH2SO—, by methods known in the art and further described in thefollowing references: Spatola, A. F. in “Chemistry and Biochemistry ofAmino Acids, Peptides, and Proteins” Weinstein, B., ed., Marcel Dekker,New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1,Issue 3, “Peptide Backbone Modifications” (general review); Morley, J.S. (1980) Trends Pharm. Sci. pp. 463-468 (general review); Hudson, D. etal. (1979) Int. J. Pept. Prot. Res. 14:177-185 (—CH2NH—, CH2CH2-);Spatola, A. F. et al. (1986) Life Sci. 38:1243-1249 (—CH2-S); Hann, M.M. (1982) J. Chem. Soc. Perkin Trans. I. 307-314 (—CH—CH—, cis andtrans); Almquist, R. G. et al. (190) J. Med. Chem. 23:1392-1398(—COCH2-); Jennings-White, C. et al. (1982) Tetrahedron Lett. 23:2533(—COCH2-); Szelke, M. et al. European Appln. EP 45665 (1982) CA:97:39405 (1982)(—CH(OH)CH2-); Holladay, M. W. et al. (1983) TetrahedronLett. (1983) 24:4401-4404 (—C(OH)CH2-); and Hruby, V. J. (1982) LifeSci. (1982) 31:189-199 (—CH2-S—); each of which is incorporated hereinby reference. A particularly preferred nonpeptide linkage is —CH2NH—.Such peptide mimetics may have significant advantages over polypeptideembodiments, including, for example: more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers. Labeling of peptidomimetics usually involves covalent attachmentof one or more labels, directly or through a spacer (e.g., an amidegroup), to non-interfering position(s) on the peptidomimetic that arepredicted by quantitative structure-activity data and/or molecularmodeling. Such non-interfering positions generally are positions that donot form direct contacts with the macropolypeptides(s) to which thepeptidomimetic binds to produce the therapeutic effect. Derivitization(e.g., labeling) of peptidomimetics should not substantially interferewith the desired biological or pharmacological activity of thepeptidomimetic.

Also encompassed by the present invention are small molecules which canmodulate (either enhance or inhibit) interactions, e.g., theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof. The small moleculesof the present invention can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. (Lam, K. S. (1997) Anticancer DrugDes. 12: 145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gal1 op et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds can be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.). Compounds can be screened in cell based or non-cell basedassays. Compounds can be screened in pools (e.g. multiple compounds ineach testing sample) or as individual compounds. In one embodiment, thesmall molecule binds to the binding site involved in interactionsbetween a Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof.

The invention also relates to Gal1 chimeric or fusion proteins. As usedherein, a Gal1 “chimeric protein” or “fusion protein” comprises a Gal1molecule or a fragment thereof operatively linked to a non-Gal1molecule. A “Gal1 molecule” refers to a polypeptide having an amino acidsequence corresponding to Gal1 or a fragment thereof, whereas a “anon-Gal1 molecule” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially homologous to therespective Gal1 molecule, e.g., a protein which is different from theGal1 molecule, and which is derived from the same or a differentorganism. Within a Gal1 fusion protein, the Gal1 portion can correspondto all or a portion of a full length Gal1 molecule. In a preferredembodiment, the fusion protein comprises at least one biologicallyactive portion of a Gal1 molecule, e.g., the carbohydrate recognitiondomain (CRD). Within the fusion protein, the term “operatively linked”is intended to indicate that the Gal1 sequences and the non-Gal1polypeptide sequences are fused in-frame to each other in such a way asto preserve functions exhibited when expressed independently of thefusion. The non-Gal1 sequences can be fused to the N-terminus orC-terminus of the Gal1 sequences, respectively.

Such a fusion protein can be produced by recombinant expression of anucleotide sequence encoding the first peptide and a nucleotide sequenceencoding the second peptide. The second peptide may optionallycorrespond to a moiety that alters the solubility, affinity, stabilityor valency of the first peptide, for example, an immunoglobulin constantregion. Preferably, the first peptide consists of a portion of Gal1 thatcomprises at least one biologically active portion of a Gal1 molecule,e.g., the carbohydrate recognition domain (CRD). In another preferredembodiment, the first peptide consists of a portion of a biologicallyactive molecule (e.g. the extracellular portion of the polypeptide orthe ligand binding portion). The second peptide can include animmunoglobulin constant region, for example, a human Cγ1 domain or Cγ4domain (e.g., the hinge, CH2 and CH3 regions of human IgCγ1, or humanIgCγ4, see e.g., Capon et al. U.S. Pat. Nos. 5,116,964; 5,580,756;5,844,095 and the like, incorporated herein by reference). Such constantregions may retain regions which mediate effector function (e.g. Fcreceptor binding) or may be altered to reduce effector function. Aresulting fusion protein may have altered solubility, binding affinity,stability and/or valency (i.e., the number of binding sites availableper polypeptide) as compared to the independently expressed firstpeptide, and may increase the efficiency of protein purification. Fusionproteins and peptides produced by recombinant techniques can be secretedand isolated from a mixture of cells and medium containing the proteinor peptide. Alternatively, the protein or peptide can be retainedcytoplasmically and the cells harvested, lysed and the protein isolated.A cell culture typically includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.Protein and peptides can be isolated from cell culture media, hostcells, or both using techniques known in the art for purifying proteinsand peptides. Techniques for transfecting host cells and purifyingproteins and peptides are known in the art.

Preferably, a fusion protein of the invention is produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). A polypeptide encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the Gal1 encoding sequences.

In another embodiment, the fusion protein contains a heterologous signalsequence at its N-terminus. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a polypeptide can be increasedthrough use of a heterologous signal sequence.

The fusion proteins of the invention can be used as immunogens toproduce antibodies in a subject. Such antibodies may be used to purifythe respective natural polypeptides from which the fusion proteins weregenerated, or in screening assays to identify polypeptides which inhibitthe interactions between a Gal1 polypeptide or a fragment thereof andits natural binding partner(s) or a fragment(s) thereof.

Also provided herein are compositions comprising one or more nucleicacids comprising or capable of expressing at least 1, 2, 3, 4, 5, 10, 20or more small nucleic acids or antisense oligonucleotides or derivativesthereof, wherein said small nucleic acids or antisense oligonucleotidesor derivatives thereof in a cell specifically hybridize (e.g., bind)under cellular conditions, with cellular nucleic acids (e.g., Gal1 mRNAor a fragment thereof). In one embodiment, expression of the smallnucleic acids or antisense oligonucleotides or derivatives thereof in acell can enhance or upregulate one or more biological activitiesassociated with the corresponding wild-type, naturally occurring, orsynthetic small nucleic acids. In another embodiment, expression of thesmall nucleic acids or antisense oligonucleotides or derivatives thereofin a cell can inhibit expression or biological activity of cellularnucleic acids and/or proteins, e.g., by inhibiting transcription,translation and/or small nucleic acid processing of, for example, theGal1 gene or gene products or fragment(s) thereof. In one embodiment,the small nucleic acids or antisense oligonucleotides or derivativesthereof are small RNAs (e.g., microRNAs) or complements of small RNAs.In another embodiment, the small nucleic acids or antisenseoligonucleotides or derivatives thereof can be single or double strandedand are at least six nucleotides in length and are less than about 1000,900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22,21, 20, 19, 18, 17, 16, 15, or 10 nucleotides in length. In anotherembodiment, a composition may comprise a library of nucleic acidscomprising or capable of expressing small nucleic acids or antisenseoligonucleotides or derivatives thereof, or pools of said small nucleicacids or antisense oligonucleotides or derivatives thereof. A pool ofnucleic acids may comprise about 2-5, 5-10, 10-20, 10-30 or more nucleicacids comprising or capable of expressing small nucleic acids orantisense oligonucleotides or derivatives thereof.

In one embodiment, binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix. In general, “antisense” refers to the range of techniquesgenerally employed in the art, and includes any process that relies onspecific binding to oligonucleotide sequences.

Small nucleic acid and/or antisense constructs of the methods andcompositions presented herein can be delivered, for example, as anexpression plasmid which, when transcribed in the cell, produces RNAwhich is complementary to at least a unique portion of cellular nucleicacids (e.g., small RNAs, mRNA, and/or genomic DNA). Alternatively, smallnucleic acids and/or antisense constructs are oligonucleotide probesthat are generated ex vivo and which, when introduced into the cell,results in hybridization with cellular nucleic acids (e.g., Gal1 mRNA ora fragment thereof). Such oligonucleotide probes are preferably modifiedoligonucleotides that are resistant to endogenous nucleases, e.g.,exonucleases and/or endonucleases, and are therefore stable in vivo.Exemplary nucleic acid molecules for use as small nucleic acids and/orantisense oligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense therapy have been reviewed,for example, by Van der Krol et al. (1988) BioTechniques 6:958-976; andStein et al. (1988) Cancer Res 48:2659-2668.

Antisense approaches may involve the design of oligonucleotides (eitherDNA or RNA) that are complementary to cellular nucleic acids (e.g., Gal1mRNA or a fragment thereof). Absolute complementarity is not required.In the case of double-stranded antisense nucleic acids, a single strandof the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the longer the hybridizing nucleic acid, the more base mismatches with anucleic acid (e.g., RNA) it may contain and still form a stable duplex(or triplex, as the case may be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the mRNA, e.g.,the 5′ untranslated sequence up to and including the AUG initiationcodon, should work most efficiently at inhibiting translation. However,sequences complementary to the 3′ untranslated sequences of mRNAs haverecently been shown to be effective at inhibiting translation of mRNAsas well. (Wagner, R. (1994) Nature 372:333). Therefore, oligonucleotidescomplementary to either the 5′ or 3′ untranslated, non-coding regions ofgenes could be used in an antisense approach to inhibit translation ofendogenous mRNAs. Oligonucleotides complementary to the 5′ untranslatedregion of the mRNA may include the complement of the AUG start codon.Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation but could also be used in accordancewith the methods and compositions presented herein. Whether designed tohybridize to the 5′, 3′ or coding region of cellular mRNAs, smallnucleic acids and/or antisense nucleic acids should be at least sixnucleotides in length, and can be less than about 1000, 900, 800, 700,600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. In one embodimentthese studies utilize controls that distinguish between antisense geneinhibition and nonspecific biological effects of oligonucleotides. Inanother embodiment these studies compare levels of the target nucleicacid or protein with that of an internal control nucleic acid orprotein. Additionally, it is envisioned that results obtained using theantisense oligonucleotide are compared with those obtained using acontrol oligonucleotide. It is preferred that the controloligonucleotide is of approximately the same length as the testoligonucleotide and that the nucleotide sequence of the oligonucleotidediffers from the antisense sequence no more than is necessary to preventspecific hybridization to the target sequence.

Small nucleic acids and/or antisense oligonucleotides can be DNA or RNAor chimeric mixtures or derivatives or modified versions thereof,single-stranded or double-stranded. Small nucleic acids and/or antisenseoligonucleotides can be modified at the base moiety, sugar moiety, orphosphate backbone, for example, to improve stability of the molecule,hybridization, etc., and may include other appended groups such aspeptides (e.g., for targeting host cell receptors), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al.(1987) Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No.WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al. (1988)BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon(1988), Pharm. Res. 5:539-549). To this end, small nucleic acids and/orantisense oligonucleotides may be conjugated to another molecule, e.g.,a peptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

Small nucleic acids and/or antisense oligonucleotides may comprise atleast one modified base moiety which is selected from the groupincluding but not limited to 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxytiethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Small nucleic acids and/or antisenseoligonucleotides may also comprise at least one modified sugar moietyselected from the group including but not limited to arabinose,2-fluoroarabinose, xylulose, and hexose.

Small nucleic acids and/or antisense oligonucleotides can also contain aneutral peptide-like backbone. Such molecules are termed peptide nucleicacid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et al.(1996) Proc. Natl. Acad. Sci. U.S.A. 93:14670 and in Eglom et al. (1993)Nature 365:566. One advantage of PNA oligomers is their capability tobind to complementary DNA essentially independently from the ionicstrength of the medium due to the neutral backbone of the DNA. In yetanother embodiment, small nucleic acids and/or antisenseoligonucleotides comprises at least one modified phosphate backboneselected from the group consisting of a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

In a further embodiment, small nucleic acids and/or antisenseoligonucleotides are α-anomeric oligonucleotides. An α-anomericoligonucleotide forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual b-units, the strandsrun parallel to each other (Gautier et al. (1987) Nucl. Acids Res.15:6625-6641). The oligonucleotide is a 2′-0-methylribonucleotide (Inoueet al. (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

Small nucleic acids and/or antisense oligonucleotides of the methods andcompositions presented herein may be synthesized by standard methodsknown in the art, e.g., by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988) Nucl. Acids Res. 16:3209,methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

Small nucleic acids and/or antisense oligonucleotides can be deliveredto cells in vivo. A number of methods have been developed for deliveringsmall nucleic acids and/or antisense oligonucleotides DNA or RNA tocells; e.g., antisense molecules can be injected directly into thetissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systematically.

In one embodiment, small nucleic acids and/or antisense oligonucleotidesmay comprise or be generated from double stranded small interfering RNAs(siRNAs), in which sequences fully complementary to cellular nucleicacids (e.g. mRNAs) sequences mediate degradation or in which sequencesincompletely complementary to cellular nucleic acids (e.g., mRNAs)mediate translational repression when expressed within cells. In anotherembodiment, double stranded siRNAs can be processed into single strandedantisense RNAs that bind single stranded cellular RNAs (e.g., microRNAs)and inhibit their expression. RNA interference (RNAi) is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by double-stranded RNA (dsRNA) that is homologous insequence to the silenced gene. In vivo, long dsRNA is cleaved byribonuclease III to generate 21- and 22-nucleotide siRNAs. It has beenshown that 21-nucleotide siRNA duplexes specifically suppress expressionof endogenous and heterologous genes in different mammalian cell lines,including human embryonic kidney (293) and HeLa cells (Elbashir et al.(2001) Nature 411:494-498). Accordingly, translation of a gene in a cellcan be inhibited by contacting the cell with short double stranded RNAshaving a length of about 15 to 30 nucleotides or of about 18 to 21nucleotides or of about 19 to 21 nucleotides. Alternatively, a vectorencoding for such siRNAs or short hairpin RNAs (shRNAs) that aremetabolized into siRNAs can be introduced into a target cell (see, e.g.,McManus et al. (2002) RNA 8:842; Xia et al. (2002) Nature Biotechnology20:1006; and Brummelkamp et al. (2002) Science 296:550). Vectors thatcan be used are commercially available, e.g., from OligoEngine under thename pSuper RNAi System™. An exemplary Gal1 shRNA target sequence isGCTGCCAGATGGATACGAA (SEQ ID NO: 1).

Ribozyme molecules designed to catalytically cleave cellular mRNAtranscripts can also be used to prevent translation of cellular mRNAs(e.g., Gal1 mRNA or a fragment thereof) and expression of cellularpolypeptides, or both (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al. (1990) Science247:1222-1225 and U.S. Pat. No. 5,093,246). While ribozymes that cleavemRNA at site specific recognition sequences can be used to destroycellular mRNAs, the use of hammerhead ribozymes is preferred. Hammerheadribozymes cleave mRNAs at locations dictated by flanking regions thatform complementary base pairs with the target mRNA. The sole requirementis that the target mRNA have the following sequence of two bases:5′-UG-3′. The construction and production of hammerhead ribozymes iswell known in the art and is described more fully in Haseloff andGerlach (1988) Nature 334:585-591. The ribozyme may be engineered sothat the cleavage recognition site is located near the 5′ end ofcellular mRNAs; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the methods and compositions presented herein alsoinclude RNA endoribonucleases (hereinafter “Cech-type ribozymes”) suchas the one which occurs naturally in Tetrahymena thermophila (known asthe IVS, or L-19 IVS RNA) and which has been extensively described byThomas Cech and collaborators (Zaug, et al. (1984) Science 224:574-578;Zaug, et al. (1986) Science 231:470-475; Zaug, et al. (1986) Nature324:429-433; published International patent application No. WO88/04300by University Patents Inc.; Been, et al. (1986) Cell 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The methods and compositions presented herein encompasses thoseCech-type ribozymes which target eight base-pair active site sequencesthat are present in cellular genes.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.) andshould be delivered to cells which express Gal1 genes or a fragmentthereof in vivo. A preferred method of delivery involves using a DNAconstruct “encoding” the ribozyme under the control of a strongconstitutive pol III or pol II promoter, so that transfected cells willproduce sufficient quantities of the ribozyme to destroy endogenouscellular messages and inhibit translation. Because ribozymes unlikeantisense molecules, are catalytic, a lower intracellular concentrationis required for efficiency.

Nucleic acid molecules to be used in triple helix formation for theinhibition of transcription of cellular genes (e.g., the Gal1 gene or afragment thereof) are preferably single stranded and composed ofdeoxyribonucleotides. The base composition of these oligonucleotidesshould promote triple helix formation via Hoogsteen base pairing rules,which generally require sizable stretches of either purines orpyrimidines to be present on one strand of a duplex. Nucleotidesequences may be pyrimidine-based, which will result in TAT and CGCtriplets across the three associated strands of the resulting triplehelix. The pyrimidine-rich molecules provide base complementarity to apurine-rich region of a single strand of the duplex in a parallelorientation to that strand. In addition, nucleic acid molecules may bechosen that are purine-rich, for example, containing a stretch of Gresidues. These molecules will form a triple helix with a DNA duplexthat is rich in GC pairs, in which the majority of the purine residuesare located on a single strand of the targeted duplex, resulting in CGCtriplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triplehelix formation may be increased by creating a so called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′, 3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

Small nucleic acids, antisense oligonucleotides, ribozymes, and triplehelix molecules of the methods and compositions presented herein may beprepared by any method known in the art for the synthesis of DNA and RNAmolecules. These include techniques for chemically synthesizingoligodeoxyribonucleotides and oligoribonucleotides well known in the artsuch as for example solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors whichincorporate suitable RNA polymerase promoters such as the T7 or SP6polymerase promoters. Alternatively, antisense cDNA constructs thatsynthesize antisense RNA constitutively or inducibly, depending on thepromoter used, can be introduced stably into cell lines.

Moreover, various well-known modifications to nucleic acid molecules maybe introduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule or the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages within the oligodeoxyribonucleotide backbone. One of skill inthe art will readily understand that regulatable proteins, inhibitorymutants, small nucleic acids, and antisense oligonucleotides can befurther linked to another peptide or polypeptide (e.g., a heterologouspeptide), e.g., that serves as a means of protein detection.Non-limiting examples of label peptide or polypeptide moieties usefulfor detection in the invention include, without limitation, suitableenzymes such as horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; epitope tags, such as FLAG,MYC, HA, or HIS tags; fluorophores such as green fluorescent protein;dyes; radioisotopes; digoxygenin; biotin; antibodies; polymers; as wellas others known in the art, for example, in Principles of FluorescenceSpectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition(July 1999).

The modulatory agents described herein (e.g. antibodies, smallmolecules, peptides, fusion proteins, or small nucleic acids) can beincorporated into pharmaceutical compositions and administered to asubject in vivo. The compositions may contain a single such molecule oragent or any combination of modulatory agents described herein.

III. Methods of Selecting Agents that Modulate Immune Cell Activation

Another aspect of the invention relates to methods of selecting agents(e.g., antibodies, fusion proteins, peptides, small molecules, or smallnucleic acids) which modulate an immune response by modulating theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof. Such methodsutilize screening assays, including cell based and non-cell basedassays.

In one embodiment, the invention relates to assays for screeningcandidate or test compounds which bind to, or modulate the activity of,a Gal1 polypeptide or a fragment thereof, e.g., modulate the ability ofa Gal1 polypeptide or a fragment thereof to interact with, e.g. bind to,its natural binding partner(s) or a fragment(s) thereof. In oneembodiment, a method for identifying an agent to modulate an immuneresponse entails determining the ability of the agent to modulate, e.g.enhance or inhibit, the interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof. Such agents include, without limitation, antibodies, proteins,fusion proteins and small molecules.

In one embodiment, a method for identifying an agent which enhances animmune response entails determining the ability of the candidate agentto inhibit the interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof. Inanother embodiment, a method for identifying an agent to decrease animmune response entails determining the ability of a candidate agent toenhance the interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof.

In one embodiment, an assay is a cell-based assay, comprising contactinga cell expressing a Gal1 polypeptide or a fragment thereof, with a testcompound and determining the ability of the test compound to modulate(e.g. stimulate or inhibit) the binding between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof. Determining the ability of a Gal1 polypeptide or a fragmentthereof to bind to, or interact with, a binding partner or a fragmentthereof, can be accomplished, e.g., by measuring direct binding or bymeasuring a parameter of immune cell activation.

For example, in a direct binding assay, a Gal1 polypeptide, a Gal1binding partner(s), or a fragment(s) thereof, can be coupled with aradioisotope or enzymatic label such that binding of the Gal1polypeptide or a fragment thereof to its natural binding partner(s) or afragment(s) thereof can be determined by detecting the labeled moleculein a complex. For example, a Gal1 polypeptide, a Gal1 bindingpartner(s), or a fragment(s) thereof, can be labeled with ¹²⁵I, ³⁵S,¹⁴C, or ³H, either directly or indirectly, and the radioisotope detectedby direct counting of radioemmission or by scintillation counting.Alternatively, a Gal1 polypeptide, a Gal1 binding partner(s), or afragment(s) thereof, can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound to modulate the interactions between a Gal1 polypeptide ora fragment thereof and its natural binding partner(s) or a fragment(s)thereof, without the labeling of any of the interactants. For example, amicrophysiometer can be used to detect the interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof without the labeling of either a Gal1 polypeptideor a fragment thereof and its natural binding partner(s) or afragment(s) thereof (McConnell, H. M. et al. (1992) Science257:1906-1912). As used herein, a “microphysiometer” (e.g., Cytosensor)is an analytical instrument that measures the rate at which a cellacidifies its environment using a light-addressable potentiometricsensor (LAPS). Changes in this acidification rate can be used as anindicator of the interaction between compound and receptor.

In a preferred embodiment, determining the ability of the blockingagents (e.g. antibodies, fusion proteins, peptides, or small molecules)to antagonize the interaction between a given set of polypeptides can beaccomplished by determining the activity of one or more members of theset of interacting molecules. For example, the activity of Gal1 can bedetermined by detecting induction of a cellular second messenger (e.g.,H-Ras), detecting catalytic/enzymatic activity of an appropriatesubstrate, detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., chloramphenicol acetyltransferase), or detecting a cellular response regulated by a Gal1polypeptide or a fragment thereof. Determining the ability of theblocking agent to bind to or interact with said polypeptide can beaccomplished by measuring the ability of an agent to modulate immuneresponses, for example, by detecting changes in type and amount ofcytokine secretion, changes in apoptosis or proliferation, changes ingene expression or activity associated with cellular identity, or byinterfering with the ability of said polypeptide to bind to antibodiesthat recognize a portion thereof.

Agents that block or inhibit interactions between a Gal1 polypeptide ora fragment thereof and its natural binding partner(s) or a fragment(s)thereof (e.g., blocking antibodies to a Gal1 polypeptide or a fragmentthereof) can be identified by their ability to inhibit immune cellproliferation, and/or effector function, induce apoptosis, or to induceanergy when added to an in vitro assay. For example, cells can becultured in the presence of an agent that stimulates signal transductionvia an activating receptor. A number of recognized readouts of cellactivation can be employed to measure, cell proliferation, apoptosis, oreffector function (e.g., antibody production, cytokine production,phagocytosis) in the presence of the activating agent. The ability of atest agent to block this activation can be readily determined bymeasuring the ability of the agent to effect a decrease inproliferation, increase apoptosis, or effector function being measured,using techniques known in the art.

In yet another embodiment, an assay of the present invention is acell-free assay in which a Gal1 polypeptide or a fragment thereof, e.g.a biologically active fragment thereof, is contacted with a testcompound, and the ability of the test compound to bind to thepolypeptide, or biologically active portion thereof, is determined.Binding of the test compound to a Gal1 polypeptide or a fragmentthereof, can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting the Gal1polypeptide or fragment thereof, with a Gal1 natural binding partner(s)or fragment(s) thereof, to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with the polypeptide in the assay mixture, whereindetermining the ability of the test compound to interact with thepolypeptide comprises determining the ability of the test compound topreferentially bind to the polypeptide or fragment thereof, as comparedto the binding partner.

For example, a Gal1 polypeptide or a fragment thereof and its naturalbinding partner(s) or a fragment(s) thereof can be used to form an assaymixture and the ability of a polypeptide to block this interaction canbe tested by determining the ability of a Gal1 polypeptide or a fragmentthereof to bind to the Gal1 natural binding partner(s) or a fragment(s)thereof, by one of the methods described above for determining directbinding. Determining the ability of a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof canalso be accomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA) (Sjolander, S, and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705). As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological polypeptides. A Gal1 polypeptideor a fragment thereof can be immobilized on a BIAcore chip and multipleagents, e.g., blocking antibodies, fusion proteins, peptides, or smallmolecules, can be tested for binding to the immobilized Gal1 polypeptideor fragment thereof. An example of using the BIA technology is describedby Fitz et al. (1997) Oncogene 15:613.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of proteins (e.g., Gal1polypeptides, Gal1 binding partner(s) polypeptides, and fragmentsthereof). In the case of cell-free assays in which a membrane-bound formprotein is used (e.g., a cell surface Gal1 polypeptide or a fragmentthereof or Gal1 natural binding partner(s) or a fragment(s) thereof) itmay be desirable to utilize a solubilizing agent such that themembrane-bound form of the protein is maintained in solution. Examplesof such solubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In one or more embodiments of the above described assay methods, it maybe desirable to immobilize either the Gal1 polypeptide, the Gal1 naturalbinding partner(s) polypeptide, or fragments thereof, to facilitateseparation of complexed from uncomplexed forms of one or both of theproteins, as well as to accommodate automation of the assay. Binding ofa test compound to a Gal1 polypeptide, a Gal1 natural binding partner(s)polypeptide, or fragments thereof, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/Gal1 or glutathione-S-transferase/Gal1 naturalbinding partner(s) fusion proteins, can be adsorbed onto glutathioneSepharose® beads (Sigma Chemical, St. Louis, Mo.) or glutathionederivatized microtiter plates, which are then combined with the testcompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads or microtiter plate wells are washed toremove any unbound components, the matrix immobilized in the case ofbeads, complex determined either directly or indirectly, for example, asdescribed above. Alternatively, the complexes can be dissociated fromthe matrix, and the level of Gal1 binding or activity determined usingstandard techniques.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a Gal1 or Gal1 natural bindingpartner(s) can be accomplished by determining the ability of the testcompound to modulate the expression or activity of a gene, e.g., nucleicacid, or gene product, e.g., polypeptide, that functions downstream ofGal1 or a Gal1 natural binding partner(s), e.g., a polypeptide thatfunctions downstream of the Gal1 natural binding partner(s). Forexample, levels of second messengers can be determined, the activity ofthe interactor polypeptide on an appropriate target can be determined,or the binding of the interactor to an appropriate target can bedetermined as previously described.

In another embodiment, modulators of Gal1 expression are identified in amethod wherein a cell is contacted with a candidate compound and theexpression of Gal1 mRNA or polypeptide or fragments thereof in the cellis determined. The level of expression of Gal1 mRNA or polypeptide orfragments thereof in the presence of the candidate compound is comparedto the level of expression of Gal1 mRNA or polypeptide or fragmentsthereof in the absence of the candidate compound. The candidate compoundcan then be identified as a modulator of Gal1 expression based on thiscomparison. For example, when expression of Gal1 mRNA or polypeptide orfragments thereof is greater (statistically significantly greater) inthe presence of the candidate compound than in its absence, thecandidate compound is identified as a stimulator of Gal1 expression.Alternatively, when expression of Gal1 mRNA or polypeptide or fragmentsthereof is reduced (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound isidentified as an inhibitor of Gal1 expression. The expression level ofGal1 mRNA or polypeptide or fragments thereof in the cells can bedetermined by methods described herein for detecting Gal1 mRNA orpolypeptide or fragments thereof.

In yet another aspect of the invention, Gal1 polypeptides or fragmentsthereof can be used as “bait proteins” in a two-hybrid assay orthree-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al.(1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Twabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identifyother polypeptides which bind to or interact with Gal1 or fragmentsthereof (“Gal1-binding proteins”, “Gal1 binding partners”, or “Gal1-bp”)and are involved in Gal1 activity. Such Gal1-binding proteins are alsolikely to be involved in the propagation of signals by the Gal1polypeptides or Gal1 natural binding partner(s) as, for example,downstream elements of a Gal1-mediated signaling pathway. Alternatively,such Gal1-binding polypeptides may be Gal1 inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a Gal1 polypeptideis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedpolypeptide (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” polypeptides are able to interact, in vivo, forming aGal1-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes thepolypeptide which interacts with the Gal1 polypeptide.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell-free assay, and the abilityof the agent to modulate the activity of a Gal1 polypeptide or afragment thereof can be confirmed in vivo, e.g., in an animal such as ananimal model for cellular transformation and/or tumorigenesis.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

IV. Pharmaceutical Compositions

Gal1 modulating agents (e.g., agents that inhibit or promote theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment thereof, including, e.g.,blocking antibodies, peptides, fusion proteins, or small molecules) canbe incorporated into pharmaceutical compositions suitable foradministration to a subject. Such compositions typically comprise theantibody, peptide, fusion protein or small molecule and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringeability exists. It must be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., blocking antibodies, peptides, fusion proteins, or smallmolecules that inhibit the interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, modulatory agents are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations should be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by, and directlydependent on, the unique characteristics of the active compound, theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity of Gal1 nucleic acid, polypeptide, or fragments thereof. Anagent may, for example, be a small molecule. For example, such smallmolecules include, but are not limited to, peptides, peptidomimetics,amino acids, amino acid analogs, polynucleotides, polynucleotideanalogs, nucleotides, nucleotide analogs, organic or inorganic compounds(i.e., including heterorganic and organometallic compounds) having amolecular weight less than about 10,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 5,000grams per mole, organic or inorganic compounds having a molecular weightless than about 1,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 500 grams per mole, and salts,esters, and other pharmaceutically acceptable forms of such compounds.It is understood that appropriate doses of small molecule agents dependsupon a number of factors within the scope of knowledge of the ordinarilyskilled physician, veterinarian, or researcher. The dose(s) of the smallmolecule will vary, for example, depending upon the identity, size, andcondition of the subject or sample being treated, further depending uponthe route by which the composition is to be administered, if applicable,and the effect which the practitioner desires the small molecule to haveupon the nucleic acid or polypeptide of the invention.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram). It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such polypeptides may include, for example, a toxin such asabrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a proteinsuch as tumor necrosis factor, alpha-interferon, beta-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al. “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

The above described modulating agents may be administered it the form ofexpressible nucleic acids which encode said agents. Such nucleic acidsand compositions in which they are contained, are also encompassed bythe present invention. For instance, the nucleic acid molecules of theinvention can be inserted into vectors and used as gene therapy vectors.Gene therapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The Gal1 molecules, e.g., the Gal1 nucleic acid molecules, polypeptides,polypeptide homologues, antibodies, and fragments thereof, describedherein can be used in one or more of the following methods: a) screeningassays; b) predictive medicine (e.g., diagnostic assays, prognosticassays, and monitoring clinical trials); and c) methods of treatment(e.g., therapeutic and prophylactic, e.g., by up- or down-modulating theimmune response). As described herein, a Gal1 polypeptide or fragmentthereof of the invention has one or more of the following activities: 1)binds to and/or modulates the activity of its natural bindingpartner(s), 2) modulates intra- or intercellular signaling, 3) modulatesactivation of T lymphocytes, 4) modulates the immune response of anorganism, e.g., a mammalian organism, such as a mouse or human. See, forexample, Toscano et al. (2007) Cyt Growth Fact Rev 18:57-71; Camby etal. (2006) Glycobiol 16:137R-157R, each of which is incorporated herein,by reference, in its entirety.

The isolated nucleic acid molecules of the invention can be used, forexample, to express a Gal1 polypeptide or a fragment thereof (e.g., viaa recombinant expression vector in a host cell in gene therapyapplications), to detect Gal1 mRNA or a fragment thereof (e.g., in abiological sample) or a genetic alteration in a Gal1 gene, and tomodulate Gal1 activity, as described further below. The Gal1polypeptides or fragments thereof can be used to treat conditions ordisorders characterized by insufficient or excessive production of aGal1 polypeptide or fragment thereof or production of Gal1 polypeptideinhibitors. In addition, the Gal1 polypeptides or fragments thereof canbe used to screen for naturally occurring Gal1 binding partner(s), toscreen for drugs or compounds which modulate Gal1 activity, as well asto treat conditions or disorders characterized by insufficient orexcessive production of Gal1 polypeptide or a fragment thereof orproduction of Gal1 polypeptide forms which have decreased, aberrant orunwanted activity compared to Gal1 wild-type polypeptides or fragmentsthereof (e.g., immune system disorders such as severe combinedimmunodeficiency, multiple sclerosis, systemic lupus erythematosus, typeI diabetes mellitus, lymphoproliferative syndrome, inflammatory boweldisease, allergies, asthma, graft-versus-host disease, and transplantrejection; immune responses to infectious pathogens such as bacteria andviruses; and immune system cancers such as cancers including mature BCell Neoplasms (e.g. Chronic lymphocytic leukemia/small lymphocyticlymphoma, B-cell prolymphocytic leukemia, Lymphoplasmacyticlymphoma/Waldenstrom macroglobulinemia, Splenic marginal zone lymphoma,Plasma cell neoplasms, Extranodal marginal zone B cell lymphoma (MALTlymphoma), Nodal marginal zone B cell lymphoma, Follicular lymphoma,Mantle cell lymphoma, Diffuse large B cell lymphoma, Mediastinal(thymic) large B cell lymphoma, Intravascular large B cell lymphoma,Primary effusion lymphoma, Burkitt lymphoma/leukemia, Lymphomatoidgranulomatosis), Mature T cell and Natural Killer (NK) Cell Neoplasms(e.g. T cell prolymphocytic leukemia, T cell large granular lymphocyticleukemia, Aggressive NK cell leukemia, Adult T cell leukemia/lymphoma,Nasal type extranodal NK/T cell lymphoma, Enteropathy-type T celllymphoma, Hepatosplenic T cell lymphoma, Blastic NK cell lymphoma,Mycosis fungoides/Sezary syndrome, Primary cutaneous CD30-positive Tcell lymphoproliferative disorders, Angioimmunoblastic T cell lymphoma,Unspecified peripheral T cell lymphoma, Anaplastic large cell lymphoma),Hodgkin lymphoma (e.g. Nodular lymphocyte-predominant Hodgkin lymphoma,Classical Hodgkin lymphoma), Immunodeficiency-AssociatedLymphoproliferative Disorders (i.e. those associated with a primaryimmune disorder, those associated with the Human Immunodeficiency Virus(HIV), those associated with Methotrexate therapy, those associated withorgan transplantation), Histiocytic and Dendritic Cell Neoplasms (e.g.Histiocytic sarcoma, Langerhans cell histiocytosis, Langerhans cellsarcoma, Interdigitating dendritic cell sarcoma/tumour, Folliculardendritic cell sarcoma/tumour, Unspecified dendritic cell sarcoma),thymomas, hamartomatous tumors arising in a vascular organ (e.g. lung,liver, uterus, hypothalmus, kidney, spleen, skeletal muscle, abdominalwall, adrenal gland, bone marrow, omentum, lymph node, skin), malignantangiosarcomas derived from such hamartomatous tumors, hemangiomas,Cowden syndrome, Lhermitte-Duclos disease, and Bannayan-Zonanasyndrome). Moreover, the anti-Gal1 antibodies or fragments thereof ofthe invention can be used to detect and isolate Gal1 polypeptides orfragments thereof, regulate the bioavailability of Gal1 polypeptides orfragments thereof, and modulate Gal1 activity, e.g., by modulating theinteraction between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof.

A. Screening Assays

In one aspect, the invention relates to a method for preventing in asubject, a disease or condition associated with an unwanted or less thandesirable immune response. Subjects at risk for a disease that wouldbenefit from treatment with the claimed agents or methods can beidentified, for example, by any or a combination of diagnostic orprognostic assays known in the art and described herein (see, forexample, agents and assays described in III. Methods of Selecting Agentsthat Modulate Immune Cell Activation).

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the Gal1 nucleotide sequences, describedherein, can be used to map the location of the Gal1 genes on achromosome. The mapping of the Gal1 sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, Gal1 genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the Gal1 nucleotidesequences. Computer analysis of the Gal1 sequences can be used topredict primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the Gal1 sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme will be retained. By using various media, panels of hybridcell lines can be established. Each cell line in a panel contains eithera single human chromosome or a small number of human chromosomes, and afull set of mouse chromosomes, allowing easy mapping of individual genesto specific human chromosomes (D'Eustachio, P. et al. (1983) Science220:919-924). Somatic cell hybrids containing only fragments of humanchromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the Gal1nucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a Gal1sequence to its chromosome include in situ hybridization (described inFan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results in a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridization during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data (such data are found, for example, in McKusick,V., Mendelian Inheritance in Man, available online through Johns HopkinsUniversity Welch Medical Library). The relationship between a gene and adisease, mapped to the same chromosomal region, can then be identifiedthrough linkage analysis (co-inheritance of physically adjacent genes),described in, for example, Egeland, J. et al. (1987) Nature 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the Gal1 gene can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The Gal1 sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the Gal1 nucleotide sequences described herein can be usedto prepare two PCR primers from the 5′ and 3′ ends of the sequences.These primers can then be used to amplify an individual's DNA andsubsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The Gal1 nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of Gal1 can providepositive individual identification with a panel of perhaps 10 to 1,000primers which each yield a noncoding amplified sequence of 100 bases. Ifpredicted Gal1 coding sequences are used, a more appropriate number ofprimers for positive individual identification would be 500-2000.

If a panel of reagents from Gal1 nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Gal1 Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of Gal1 are particularly appropriate forthis use as greater numbers of polymorphisms occur in the noncodingregions, making it easier to differentiate individuals using thistechnique. Examples of polynucleotide reagents include the Gal1nucleotide sequences or portions thereof, e.g., fragments derived fromthe noncoding regions of Gal1 having a length of at least 20 bases,preferably at least 30 bases.

The Gal1 nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., lymphocytes. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such Gal1 probes can be used to identifytissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., Gal1 primers or probes canbe used to screen tissue culture for contamination (i.e., screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining Gal1 polypeptideand/or nucleic acid expression as well as Gal1 activity, in the contextof a biological sample (e.g., blood, serum, cells, or tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant orunwanted Gal1 expression or activity. The invention also provides forprognostic (or predictive) assays for determining whether an individualis at risk of developing a disorder associated with Gal1 polypeptide,nucleic acid expression or activity. For example, mutations in a Gal1gene can be assayed in a biological sample.

Such assays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with Gal1 polypeptide, nucleic acidexpression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of Gal1 inclinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of Gal1polypeptide or nucleic acid or fragments thereof in a biological sampleinvolves obtaining a biological sample from a test subject andcontacting the biological sample with a compound or an agent capable ofdetecting Gal1 polypeptide or nucleic acid that encodes Gal1 polypeptide(e.g., mRNA or genomic DNA) or fragments thereof such that the presenceof Gal1 polypeptide or nucleic acid or fragments thereof is detected inthe biological sample. A preferred agent for detecting Gal1 mRNA,genomic DNA, or fragments thereof is a labeled nucleic acid probecapable of hybridizing to Gal1 mRNA, genomic DNA, or fragments thereof.The nucleic acid probe can be, for example, full length Gal1 nucleicacid, or a portion thereof, such as an oligonucleotide of at least 15,30, 50, 100, 250 or 500 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to Gal1 mRNA orgenomic DNA. Other suitable probes for use in the diagnostic assays ofthe invention are described herein.

A preferred agent for detecting a Gal1 polypeptide or a fragment thereofis an antibody capable of binding to a Gal1 polypeptide, preferably anantibody with a detectable label. Antibodies can be polyclonal, or morepreferably, monoclonal. An intact antibody, or a fragment thereof (e.g.,Fab or F(ab′)2) can be used. The term “labeled”, with regard to theprobe or antibody, is intended to encompass direct labeling of the probeor antibody by coupling (i.e., physically linking) a detectablesubstance to the probe or antibody, as well as indirect labeling of theprobe or antibody by reactivity with another reagent that is directlylabeled. Examples of indirect labeling include detection of a primaryantibody using a fluorescently labeled secondary antibody andend-labeling of a DNA probe with biotin such that it can be detectedwith fluorescently labeled streptavidin. The term “biological sample” isintended to include tissues, cells, and biological fluids isolated froma subject, as well as tissues, cells, and fluids present within asubject. That is, the detection method of the invention can be used todetect Gal1 mRNA, polypeptide, genomic DNA, or fragments thereof, in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of Gal1 mRNA or a fragment thereof includeNorthern hybridizations and in situ hybridizations. In vitro techniquesfor detection of Gal1 polypeptide include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of Gal1 genomicDNA or a fragment thereof include Southern hybridizations. Furthermore,in vivo techniques for detection of a Gal1 polypeptide or a fragmentthereof include introducing into a subject a labeled anti-Gal1 antibody.For example, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

In one embodiment, the biological sample contains polypeptide moleculesfrom the test subject. Alternatively, the biological sample can containmRNA molecules from the test subject or genomic DNA molecules from thetest subject. A preferred biological sample is a serum sample isolatedby conventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting Gal1 polypeptide, mRNA,genomic DNA, or fragments thereof, such that the presence of Gal1polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in thebiological sample, and comparing the presence of Gal1 polypeptide, mRNA,genomic DNA, or fragments thereof, in the control sample with thepresence of Gal1 polypeptide, mRNA, genomic DNA, or fragments thereof inthe test sample.

The invention also encompasses kits for detecting the presence of a Gal1nucleic acid, polypeptide, or fragments thereof, in a biological sample.For example, the kit can comprise a labeled compound or agent capable ofdetecting a Gal1 nucleic acid, polypeptide, or fragments thereof in abiological sample; means for determining the amount of the Gal1 nucleicacid, polypeptide, or fragments thereof in the sample; and means forcomparing the amount of the Gal1 nucleic acid, polypeptide, or fragmentsthereof in the sample with a standard. The compound or agent can bepackaged in a suitable container. The kit can further compriseinstructions for using the kit to detect the Gal1 nucleic acid,polypeptide, or fragments thereof.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted Gal1 expression or activity. Asused herein, the term “aberrant” includes a Gal1 expression or activitywhich deviates from the wild type Gal1 expression or activity. Aberrantexpression or activity includes increased or decreased expression oractivity, as well as expression or activity which does not follow thewild type developmental pattern of expression or the subcellular patternof expression. For example, aberrant Gal1 expression or activity isintended to include the cases in which a mutation in the Gal1 gene orregulatory sequence thereof causes the Gal1 gene to be under-expressedor over-expressed and situations in which such mutations result in anon-functional Gal1 polypeptide or a polypeptide which does not functionin a wild-type fashion, e.g., a polypeptide which does not interact witha Gal1 binding partner(s) or one which interacts with a non-Gal1 bindingpartner(s). As used herein, the term “unwanted” includes an unwantedphenomenon involved in a biological response such as immune cellactivation. For example, the term unwanted includes a Gal1 expression oractivity which is undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in Gal1polypeptide activity or nucleic acid expression, such as an autoimmunedisorder, an immunodeficiency disorder, an immune system cancer, e.g.,Hodgkin lymphoma, or a tendency to have spontaneous abortions.Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing a disorder associated with amisregulation of Gal1 polypeptide activity or nucleic acid expression,such as an autoimmune disorder, and immunodeficiency disorder, or animmune system cancer, e.g., Hodgkin lymphoma. Thus, the presentinvention provides a method for identifying a disease or disorderassociated with aberrant or unwanted Gal1 expression or activity inwhich a test sample is obtained from a subject and Gal1 polypeptide ornucleic acid (e.g., mRNA or genomic DNA) is detected, wherein thepresence of Gal1 polypeptide or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted Gal1 expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,cerebrospinal fluid or serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted Gal1 expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for an autoimmune disorder,immunodeficiency disorder, or immune system cancer, e.g., Hodgkinlymphoma. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant or unwanted Gal1 expression oractivity in which a test sample is obtained and Gal1 polypeptide ornucleic acid expression or activity is detected (e.g., wherein theabundance of Gal1 polypeptide or nucleic acid expression or activity isdiagnostic for a subject that can be administered the agent to treat adisorder associated with aberrant or unwanted Gal1 expression oractivity).

The methods of the invention can also be used to detect geneticalterations in a Gal1 gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inGal1 polypeptide activity or nucleic acid expression, such as anautoimmune disorder, an immunodeficiency disorder, or an immune systemcancer, e.g., Hodgkin lymphoma. In preferred embodiments, the methodsinclude detecting, in a sample of cells from the subject, the presenceor absence of a genetic alteration characterized by at least onealteration affecting the integrity of a gene encoding a Gal1polypeptide, or the mis-expression of the Gal1 gene. For example, suchgenetic alterations can be detected by ascertaining the existence of atleast one of 1) a deletion of one or more nucleotides from a Gal1 gene,2) an addition of one or more nucleotides to a Gal1 gene, 3) asubstitution of one or more nucleotides of a Gal1 gene, 4) a chromosomalrearrangement of a Gal1 gene, 5) an alteration in the level of amessenger RNA transcript of a Gal1 gene, 6) aberrant modification of aGal1 gene, such as of the methylation pattern of the genomic DNA, 7) thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of a Gal1 gene, 8) a non-wild type level of a Gal1polypeptide, 9) allelic loss of a Gal1 gene, and 10) inappropriatepost-translational modification of a Gal1 polypeptide. As describedherein, there are a large number of assays known in the art which can beused for detecting alterations in a Gal1 gene. A preferred biologicalsample is a tissue or serum sample isolated by conventional means from asubject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a Gal1 gene (seeAbravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a Gal1 gene under conditions such thathybridization and amplification of the Gal1 gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a Gal1 gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in Gal1 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotideprobes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J.et al. (1996) Nat. Med. 2:753-759). For example, genetic mutations inGal1 can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin et al. (1996) supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequential,overlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the Gal1 gene anddetect mutations by comparing the sequence of the sample Gal1 with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or Sanger (1977) Proc.Natl. Acad. Sci. USA 74:5463. It is also contemplated that any of avariety of automated sequencing procedures can be utilized whenperforming the diagnostic assays (Naeve, C. W. (1995) Biotechniques19:448-53), including sequencing by mass spectrometry (see, e.g., PCTInternational Publication No. WO 94/16101; Cohen et al. (1996) Adv.Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.Biotechnol. 38:147-159).

Other methods for detecting mutations in the Gal1 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes formed by hybridizing (labeled) RNA or DNAcontaining the wild-type Gal1 sequence with potentially mutant RNA orDNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digest the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397 and Saleeba et al. (1992) MethodsEnzymol. 217:286-295. In a preferred embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in Gal1 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a Gal1 sequence,e.g., a wild-type Gal1 sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in Gal1 genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci. USA 86:2766; see also Cotton(1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control Gal1nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to ensure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell. Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a Gal1 gene.

Furthermore, any cell type or tissue in which Gal1 is expressed may beutilized in the prognostic assays described herein.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a Gal1 polypeptide or a fragment thereof (e.g., themodulation of cell proliferation and/or migration) can be applied notonly in basic drug screening, but also in clinical trials. For example,the effectiveness of an agent determined by a screening assay asdescribed herein to increase Gal1 gene expression, polypeptide levels,or upregulate Gal1 activity, can be monitored in clinical trials ofsubjects exhibiting decreased Gal1 gene expression, polypeptide levels,or downregulated Gal1 activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease Gal1 gene expression,polypeptide levels, or downregulate Gal1 activity, can be monitored inclinical trials of subjects exhibiting increased Gal1 gene expression,polypeptide levels, or Gal1 activity. In such clinical trials, theexpression or activity of a Gal1 gene, and preferably, other genes thathave been implicated in, for example, a Gal1-associated disorder can beused as a “read out” or marker of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including Gal1, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates Gal1 activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on Gal1-associated disorders (e.g., disorderscharacterized by dysregulated Gal1 activity), for example, in a clinicaltrial, cells can be isolated and RNA prepared and analyzed for thelevels of expression of Gal1 and other genes implicated in theGal1-associated disorder, respectively. The levels of gene expression(e.g., a gene expression pattern) can be quantified by Northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of polypeptide produced, by one of the methods as describedherein, or by measuring the levels of activity of Gal1 or other genes.In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide,nucleic acid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a Gal1 polypeptide,mRNA, genomic DNA, or fragments thereof in the preadministration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression or activity of the Gal1polypeptide, mRNA, genomic DNA, or fragments thereof in thepost-administration samples; (v) comparing the level of expression oractivity of the Gal1 polypeptide, mRNA, genomic DNA, or fragmentsthereof in the pre-administration sample with the Gal1 polypeptide,mRNA, or genomic DNA in the post administration sample or samples; and(vi) altering the administration of the agent to the subjectaccordingly. For example, increased administration of the agent may bedesirable to increase the expression or activity of Gal1 to higherlevels than detected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of Gal1 to lower levels than detected,i.e., to decrease the effectiveness of the agent. According to such anembodiment, Gal1 expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observablephenotypic response.

D. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disordercharacterized by insufficient or excessive production of Gal1polypeptides or production of Gal1 protein forms which have decreased oraberrant activity compared to Gal1 wild type protein. Moreover, theanti-Gal1 antibodies of the invention can be used to detect and isolateGal1 polypeptides or fragments thereof, regulate the bioavailability ofGal1 polypeptides or fragments thereof, and modulate Gal1 activity e.g.,by modulating the interaction of a Gal1 polypeptide or a fragmentthereof with its natural binding partner(s) or fragments(s) thereof.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedGal1 expression or activity, by administering to the subject a Gal1polypeptide or a fragment thereof or an agent which modulates Gal1expression or at least one Gal1 activity. Subjects at risk for a diseaseor disorder which is caused or contributed to by aberrant or unwantedGal1 expression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the Gal1 aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of Gal1 aberrancy, for example, aGal1 polypeptide, Gal1 agonist or Gal1 antagonist (e.g., an anti-Gal1antibody or a combination of anti-Gal1 and antibodies against otherimmune related targets) agent can be used for treating the subject. Theappropriate agent can be determined based on screening assays describedherein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating Gal1expression or activity or interaction with its natural bindingpartner(s), for therapeutic purposes. Gal1 has been demonstrated toinhibit the viability of CD3⁺ cells and CD4⁺ T cells and contribute to aTh1/Th2 imbalance. Accordingly, the activity and/or expression of Gal1,as well as the interaction between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof canbe modulated in order to modulate the immune response.

Modulatory methods of the invention involve contacting a cell with aGal1 polypeptide or a fragment thereof or agent that modulates one ormore of the activities of Gal1 polypeptide activity associated with thecell, e.g., an agent that modulates expression or activity of Gal1and/or modulates the interaction of a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof. Anagent that modulates Gal1 polypeptide activity can be an agent asdescribed herein, such as a nucleic acid or a polypeptide, anaturally-occurring binding partner of a Gal1 polypeptide, a Gal1antibody, a combination of Gal1 antibodies and antibodies against otherimmune related targets, a Gal1 agonist or antagonist, a peptidomimeticof a Gal1 agonist or antagonist, a Gal1 peptidomimetic, other smallmolecule, or small RNA directed against a Gal1 nucleic acid geneexpression product.

An agent that modulates the expression of Gal1 is, e.g., an antisensenucleic acid molecule, RNAi molecule, shRNA or other small RNA molecule,triplex oligonucleotide, ribozyme, or recombinant vector for expressionof a Gal1 polypeptide. For example, an oligonucleotide complementary tothe area around a Gal1 polypeptide translation initiation site can besynthesized. One or more antisense oligonucleotides can be added to cellmedia, typically at 200 μg/ml, or administered to a patient to preventthe synthesis of a Gal1 polypeptide. The antisense oligonucleotide istaken up by cells and hybridizes to a Gal1 mRNA to prevent translation.Alternatively, an oligonucleotide which binds double-stranded DNA toform a triplex construct to prevent DNA unwinding and transcription canbe used. As a result of either, synthesis of Gal1 polypeptide isblocked. When Gal1 expression is modulated, preferably, such modulationoccurs by a means other than by knocking out the Gal1 gene.

Agents which modulate expression, by virtue of the fact that theycontrol the amount of Gal1 in a cell, also modulate the total amount ofGal1 activity in a cell.

In one embodiment, the agent the modulates Gal1 stimulates one or moreGal1 activities. Examples of such stimulatory agents include active Gal1polypeptide or a fragment thereof and a nucleic acid molecule encodingGal1 or a fragment thereof that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more Gal1 activities. In apreferred embodiment, the agent inhibits or enhances the interaction ofGal1 with its natural binding partner(s). Examples of such inhibitoryagents include antisense Gal1 nucleic acid molecules, anti-Gal1antibodies, Gal1 inhibitors, and compounds identified in the subjectscreening assays.

These modulatory methods can be performed in vitro (e.g., by contactingthe cell with the agent) or, alternatively, by contacting an agent withcells in vivo (e.g., by administering the agent to a subject). As such,the present invention provides methods of treating an individualafflicted with a condition or disorder that would benefit from up- ordownmodulation of a Gal1 polypeptide or a fragment thereof, e.g., adisorder characterized by unwanted, insufficient, or aberrant expressionor activity of a Gal1 polypeptide or nucleic acid molecule or fragmentsthereof. In one embodiment, the method involves administering an agent(e.g., an agent identified by a screening assay described herein), orcombination of agents that modulates (e.g., upregulates ordownregulates) Gal1 expression or activity. In another embodiment, themethod involves administering a Gal1 polypeptide or nucleic acidmolecule as therapy to compensate for reduced, aberrant, or unwantedGal1 expression or activity.

Stimulation of Gal1 activity is desirable in situations in which Gal1 isabnormally downregulated and/or in which increased Gal1 activity islikely to have a beneficial effect. Likewise, inhibition of Gal1activity is desirable in situations in which Gal1 is abnormallyupregulated and/or in which decreased Gal1 activity is likely to have abeneficial effect.

Exemplary agents for use in downmodulating Gal1 (i.e., Gal1 antagonists)include, e.g., antisense nucleic acid molecules, antibodies thatrecognize and block Gal1, combinations of antibodies that recognize andblock Gal1 and antibodies that recognize and block other immune relatedtargets, and compounds that block the interaction of a Gal1 polypeptideor a fragment thereof with its naturally occurring binding partner(s) orfragment(s) thereof on an immune cell. Exemplary agents for use inupmodulating Gal1 (i.e., Gal1 agonists) include, e.g., nucleic acidmolecules encoding Gal1 polypeptides, multivalent forms of Gal1,compounds that increase the expression of Gal1, compounds that enhancethe interaction of Gal1 with its naturally occurring binding partner(s)and cells that express Gal1.

In addition, these modulatory agents can also be administered incombination therapy with, e.g., chemotherapeutic agents, hormones,antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy,and/or radiotherapy. The preceding treatment methods can be administeredin conjunction with other forms of conventional therapy, eitherconsecutively with, pre- or post-conventional therapy. For example,these modulatory agents can be administered with a therapeuticallyeffective dose of chemotherapeutic agent. In another embodiment, thesemodulatory agents are administered in conjunction with chemotherapy toenhance the activity and efficacy of the chemotherapeutic agent. ThePhysicians' Desk Reference (PDR) discloses dosages of chemotherapeuticagents that have been used in the treatment of various cancers. Thedosing regiment and dosages of these aforementioned chemotherapeuticdrugs that are therapeutically effective will depend on the particularimmune disorder, e.g., Hodgkin lymphoma, being treated, the extent ofthe disease and other factors familiar to the physician of skill in theart and can be determined by the physician.

3. Downregulation of Immune Responses

There are numerous embodiments of the invention for modulating theinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof to therebydownregulate downregulate immune responses. Downregulation can be in theform of inhibiting or blocking an immune response already in progress,or may involve preventing the induction of an immune response. Thefunctions of activated immune cells can be inhibited by down-regulatingimmune cell responses, or by inducing specific anergy in immune cells,or both.

For example, the immune response can be downmodulated and/or anergy canbe induced using, for example, Gal1 polypeptides (e.g., soluble forms ofGal1 or Gal1 fusion polypeptides) and agents that promote binding of aGal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof (e.g., Gal1 peptide mimetics),identified by the methods described herein.

In one embodiment, fusion proteins comprising a first Gal1 peptide fusedto a second peptide can be used to enhance the interaction of a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof on an immune cell, to thereby downmodulate immuneresponses. In one embodiment, the second peptide blocks an activity ofanother immune related antigen to further downmodulate immune responses.Alternatively, two separate agents that downmodulate immune responsescan be combined as a single composition or administered separately(simultaneously or sequentially) to more effectively downregulate immunecell mediated immune responses in a subject. For instance, a Gal1polypeptide can be combined with a B7 polypeptide, or with a combinationof blocking antibodies (e.g., antibodies against Gal1 polypeptide withanti-B7-1 and/or anti-B7-2 monoclonal antibodies). Furthermore, atherapeutically active amount of one or more of the subject agents, canbe used in conjunction with other downmodulating reagents to influenceimmune responses. Examples of other immunomodulating reagents include,without limitation, antibodies that block a costimulatory signal, (e.g.,against CD28 or ICOS), antibodies that act as agonists of CTLA4, and/orantibodies against other immune cell markers (e.g., against CD40,against CD40 ligand, or against cytokines), fusion proteins (e.g.,CTLA4-Fc), immunosuppressive drugs, (e.g., rapamycin, cyclosporine A,FK506, etc.), or chemotherapy drugs (e.g., adriamycin, bleomycin,vinblastine, dacarbazine, mechloretamine, vincristine, prednisone,procarbazine, etc.).

The Gal1 polypeptides may also be useful in the construction oftherapeutic agents which block immune cell function by destruction ofcells. For example, portions of a Gal1 polypeptide can be linked to atoxin to make a cytotoxic agent capable of triggering the destruction ofcells to which it binds.

For making cytotoxic agents, polypeptides of the invention may belinked, or operatively attached, to toxins using techniques that areknown in the art, e.g., via crosslinking or recombinant DNA techniques.The preparation of immunotoxins is, in general, well known in the art(see, e.g., U.S. Pat. No. 4,340,535 and EP 44167, both incorporatedherein by reference). Numerous types of disulfide bond-containinglinkers are known which can successfully be employed to conjugate thetoxin moiety with a polypeptide. In one embodiment, linkers that containa disulfide bond that is sterically “hindered” are to be preferred, dueto their greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action.

A wide variety of toxins are known that may be conjugated topolypeptides or antibodies of the invention. Examples include: numeroususeful plant-, fungus- or even bacteria-derived toxins, which, by way ofexample, include: various A chain toxins, particularly ricin A chain;ribosome inactivating proteins such as saporin or gelonin; alpha-sarcin;aspergillin; restrictocin; and ribonucleases such as placentalribonuclease, angiogenic, diphtheria toxin, or pseudomonas exotoxin. Apreferred toxin moiety for use in connection with the invention is toxinA chain which has been treated to modify or remove carbohydrateresidues, deglycosylated A chain. (U.S. Pat. No. 5,776,427).

Upregulating or enhancing interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof is useful to downmodulate the immune response, e.g., insituations of tissue, skin and organ transplantation, ingraft-versus-host disease (GVHD), or in autoimmune diseases such assystemic lupus erythematosus, and multiple sclerosis. For example,blockage of immune cell function results in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign byimmune cells, followed by an immune reaction that destroys thetransplant. The administration of a polypeptide which enhancesinteractions between Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof, alone or inconjunction with another downmodulatory agent, prior to or at the timeof transplantation can promote the generation of an downregulated immuneresponse. Moreover, enhancement of interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof may also be sufficient to anergize the immunecells, thereby inducing tolerance in a subject. Induction of long-termtolerance by enhancing interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof may avoid the necessity of repeated administration of theseblocking reagents.

To achieve sufficient immunosuppression or tolerance in a subject, itmay also be desirable to block the costimulatory function of otherpolypeptides. For example, it may be desirable to block the function ofB7-1, B7-2, or B7-1 and B7-2 by administering a soluble form of acombination of peptides having an activity of each of these antigens,blocking antibodies against these antigens or blocking small molecules(separately or together in a single composition) prior to or at the timeof transplantation. Alternatively, it may be desirable to promoteinhibitory activity of a PD-1 ligand or PD-1 and inhibit a costimulatoryactivity of B7-1 and/or B7-2. Other downmodulatory agents that can beused in connection with the downmodulatory methods of the inventioninclude, for example, agents that transmit an inhibitory signal viaCTLA4, soluble forms of CTLA4, antibodies that activate an inhibitorysignal via CTLA4, blocking antibodies against other immune cell markersor soluble forms of other receptor ligand pairs (e.g., agents thatdisrupt the interaction between CD40 and CD40 ligand (e.g., anti CD40ligand antibodies)), antibodies against cytokines, or immunosuppressivedrugs.

Downmodulation of immune responses are also useful in treatingautoimmune disease. Many autoimmune disorders are the result ofinappropriate activation of immune cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive immune cells may reduce or eliminate disease symptoms.Administration of reagents which enhance interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof are useful for inhibiting immune cell activationand preventing production of autoantibodies or cytokines which may beinvolved in the disease process. The efficacy of reagents in preventingor alleviating autoimmune disorders can be determined using a number ofwell-characterized animal models of human autoimmune diseases. Examplesinclude murine experimental autoimmune encephalitis, systemic lupuserythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmunecollagen arthritis, diabetes mellitus in NOD mice and BB rats, andmurine experimental myasthenia gravis (see, e.g., Paul ed., FundamentalImmunology, Raven Press, New York, Third Edition 1993, chapter 30).

Inhibition of immune cell activation is useful therapeutically in thetreatment of allergy and allergic reactions, e.g., by inhibiting IgEproduction. An agent that promotes interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof can be administered to an allergic subject toinhibit immune cell mediated allergic responses in the subject. Allergicreactions can be systemic or local in nature, depending on the route ofentry of the allergen and the pattern of deposition of IgE on mast cellsor basophils. Thus, inhibition of immune cell mediated allergicresponses locally or systemically by tailored administration of an agentthat promotes interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof.

Inhibition of immune cell activation through enhancement of interactionsbetween a Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof may also be importanttherapeutically in viral infections of immune cells. For example, in theacquired immune deficiency syndrome (AIDS), viral replication isstimulated by immune cell activation. Modulation of these interactionsmay result in inhibition of viral replication and thereby ameliorate thecourse of AIDS.

In an additional embodiment, in performing any of the methods describedherein, it is within the scope of the invention to downregulate animmune response by administering one or more additional agents. Forexample, the use of other agents known to downregulate the immuneresponse can be used in conjunction with an agent that stimulates Gal1activity or interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof.

4. Upregulation of Immune Responses

Also useful therapeutically is the inhibition of interactions between aGal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof to thereby upregulate immuneresponses. Upregulation of immune responses can be in the form ofenhancing an existing immune response or eliciting an initial immuneresponse. For instance, enhancing an immune response using the subjectcompositions and methods is useful in cases of infections with microbes(e.g., bacteria, viruses, or parasites). In one embodiment, an agentthat blocks interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof isused to enhance the immune response. Such an agent (e.g., a Gal1blocking antibody) is therapeutically useful in situations whereupregulation of antibody and cell-mediated responses would bebeneficial. Exemplary disorders include cancer, especially Hodgkinlymphoma, viral skin diseases, such as Herpes or shingles, in which casesuch an agent can be delivered topically to the skin. In addition,systemic viral diseases such as influenza, the common cold, andencephalitis might be alleviated by systemic administration of suchagents.

Alternatively, immune responses can be enhanced in an infected patientthrough an ex vivo approach, for instance, by removing immune cells fromthe patient, contacting immune cells in vitro with an agent that blocksinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof, and reintroducingthe in vitro stimulated immune cells into the patient.

In certain instances, it may be desirable to further administer otheragents that upregulate immune responses, for example, forms of B7 familymembers that transduce signals via costimulatory receptors, in order tofurther augment the immune response.

An agent that inhibits Gal1 activity or interactions between a Gal1polypeptide or a fragment thereof and its natural binding partner(s) ora fragment(s) thereof, can be used prophylactically in vaccines againstvarious polypeptides, e.g., polypeptides derived from pathogens.Immunity against a pathogen, e.g., a virus, can be induced byvaccinating with a viral polypeptide along with an agent that inhibitsGal1 activity or interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof, inan appropriate adjuvant. Alternately, a vector comprising genes whichencode for both a pathogenic antigen and a form of Gal1 that blocksinteractions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof can be used forvaccination. Nucleic acid vaccines can be administered by a variety ofmeans, for example, by injection (e.g., intramuscular, intradermal, orthe biolistic injection of DNA-coated gold particles into the epidermiswith a gene gun that uses a particle accelerator or a compressed gas toinject the particles into the skin (Haynes et al. (1996) J. Biotechnol.44:37)). Alternatively, nucleic acid vaccines can be administered bynon-invasive means. For example, pure or lipid-formulated DNA can bedelivered to the respiratory system or targeted elsewhere, e.g., Peyerspatches by oral delivery of DNA (Schubbert (1997) Proc. Natl. Acad. Sci.USA 94:961). Attenuated microorganisms can be used for delivery tomucosal surfaces (Sizemore et al. (1995) Science 270:29).

In another embodiment, the antigen in the vaccine is a self-antigen.Such a vaccine is useful in the modulation of tolerance in an organism.Immunization with a self antigen and an agent that blocks Gal1 activityor interactions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof can break tolerance(i.e., interfere with tolerance of a self antigen). Such a vaccine mayalso include adjuvants such as alum or cytokines (e.g., GM-CSF, IL-12,B7-1, or B7-2).

In another embodiment, upregulation or enhancement of an immune responsefunction, as described herein, is useful in the induction of tumorimmunity (e.g., restoration of immune surveillance in Hodgkin lymphoma).Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma,or carcinoma) can be transfected with a nucleic acid molecule thatinhibits Gal1 activity or interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof. These molecules can be, e.g., nucleic acid molecules which areantisense to Gal1, or can encode non-activating anti-Gal1 antibodies orcombinations of anti-Gal1 antibodies and antibodies against other immunerelated targets. These molecules can also be the variable region of ananti-Gal1 antibody and/or an anti-Gal1 antibody. If desired, the tumorcells can also be transfected with other polypeptides which enhance animmune response. The transfected tumor cells are returned to thepatient, which results in inhibition (e.g., local inhibition) of Gal1activity or interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof.Alternatively, gene therapy techniques can be used to target a tumorcell for transfection in vivo.

Stimulation of an immune response to tumor cells can also be achieved byinhibiting Gal1 activity or interactions between a Gal1 polypeptide or afragment thereof and its natural binding partner(s) or a fragment(s)thereof, by treating a patient with an agent that inhibits Gal1 activityor interactions between a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof. Examples of suchagents include, e.g., antisense nucleic acid molecules, small RNAs,antibodies that recognize and block Gal1, a combination of antibodiesthat recognize and block Gal1 and antibodies that recognize and blockother immune related targets, compounds that block the interactionsbetween a Gal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof on an immune cell, and compoundsidentified in the subject screening assays).

In another embodiment, the immune response can be stimulated by themethods described herein, such that preexisting tolerance is overcome.For example, immune responses against antigens to which a subject cannotmount a significant immune response, e.g., to an autologous antigen,such as a tumor specific antigens can be induced by administering anagent that blocks interactions between a Gal1 polypeptide or a fragmentthereof and its natural binding partner(s) or a fragment(s) thereof. Inone embodiment, a blocking antibody that inhibits interactions between aGal1 polypeptide or a fragment thereof and its natural bindingpartner(s) or a fragment(s) thereof can be used to enhance an immuneresponse (e.g., to a tumor cell). In one embodiment, an autologousantigen, such as a tumor-specific antigen can be coadministered. Inanother embodiment, an immune response can be stimulated against anantigen (e.g., an autologous antigen) to treat an immune disorder, e.g.,Hodgkin lymphoma. In another embodiment, the subject agents can be usedas adjuvants to boost responses to foreign antigens in the process ofactive immunization.

In one embodiment, immune cells are obtained from a subject and culturedex vivo in the presence of an agent as described herein, to expand thepopulation of immune cells and/or to enhance immune cell activation. Ina further embodiment the immune cells are then administered to asubject. Immune cells can be stimulated in vitro by, for example,providing to the immune cells a primary activation signal and acostimulatory signal, as is known in the art. Various agents can also beused to costimulate proliferation of immune cells. In one embodimentimmune cells are cultured ex vivo according to the method described inPCT Application No. WO 94/29436. The costimulatory polypeptide can besoluble, attached to a cell membrane, or attached to a solid surface,such as a bead.

In an additional embodiment, in performing any of the methods describedherein, it is within the scope of the invention to upregulate an immuneresponse by administering one or more additional agents. For example,the use of other agents known to stimulate the immune response, such ascytokines, adjuvants, or stimulatory forms of costimulatory molecules ortheir ligands can be used in conjunction with an agent that inhibitsGal1 activity or a Gal1 polypeptide or a fragment thereof and itsnatural binding partner(s) or a fragment(s) thereof.

V. Administration of Agents

The immune modulating agents of the invention are administered tosubjects in a biologically compatible form suitable for pharmaceuticaladministration in vivo, to either enhance or suppress immune cellmediated immune responses. By “biologically compatible form suitable foradministration in vivo” is meant a form of the protein to beadministered in which any toxic effects are outweighed by thetherapeutic effects of the protein. The term “subject” is intended toinclude living organisms in which an immune response can be elicited,e.g., mammals. Examples of subjects include humans, dogs, cats, mice,rats, and transgenic species thereof. Administration of an agent asdescribed herein can be in any pharmacological form including atherapeutically active amount of an agent alone or in combination with apharmaceutically acceptable carrier.

Administration of a therapeutically active amount of the therapeuticcomposition of the present invention is defined as an amount effective,at dosages and for periods of time necessary, to achieve the desiredresult. For example, a therapeutically active amount of a Gal1 blockingantibody may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of peptide to elicita desired response in the individual. Dosage regimens can be adjusted toprovide the optimum therapeutic response. For example, several divideddoses can be administered daily or the dose can be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

The agents or the invention described herein can be administered in aconvenient manner such as by injection (subcutaneous, intravenous,etc.), oral administration, inhalation, transdermal application, orrectal administration. Depending on the route of administration, theactive compound can be coated in a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound. For example, for administration of agents, byother than parenteral administration, it may be desirable to coat theagent with, or co-administer the agent with, a material to prevent itsinactivation.

An agent can be administered to an individual in an appropriate carrier,diluent or adjuvant, co-administered with enzyme inhibitors or in anappropriate carrier such as liposomes. Pharmaceutically acceptablediluents include saline and aqueous buffer solutions. Adjuvant is usedin its broadest sense and includes any immune stimulating compound suchas interferon. Adjuvants contemplated herein include resorcinols,non-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatictrypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol.Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

The agent may also be administered parenterally or intraperitoneally.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof, and in oils. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

Pharmaceutical compositions of agents suitable for injectable useinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. In all cases the composition willpreferably be sterile and must be fluid to the extent that easysyringeability exists. It will preferably be stable under the conditionsof manufacture and storage and preserved against the contaminatingaction of microorganisms such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it is preferable to includeisotonic agents, for example, sugars, polyalcohols such as manitol,sorbitol, sodium chloride in the composition. Prolonged absorption ofthe injectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating an agentof the invention (e.g., an antibody, peptide, fusion protein or smallmolecule) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the agent plusany additional desired ingredient from a previously sterile-filteredsolution thereof.

When the agent is suitably protected, as described above, the proteincan be orally administered, for example, with an inert diluent or anassimilable edible carrier. As used herein “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the therapeutic compositions iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form”, as used herein, refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by, and directly dependent on, (a)the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

In one embodiment, an agent of the invention is an antibody. As definedherein, a therapeutically effective amount of antibody (i.e., aneffective dosage) ranges from about 0.001 to 30 mg/kg body weight,preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Theskilled artisan will appreciate that certain factors may influence thedosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of an antibody can include a single treatment or,preferably, can include a series of treatments. In a preferred example,a subject is treated with antibody in the range of between about 0.1 to20 mg/kg body weight, one time per week for between about 1 to 10 weeks,preferably between 2 to 8 weeks, more preferably between about 3 to 7weeks, and even more preferably for about 4, 5, or 6 weeks. It will alsobe appreciated that the effective dosage of antibody used for treatmentmay increase or decrease over the course of a particular treatment.Changes in dosage may result from the results of diagnostic assays. Inaddition, an antibody of the invention can also be administered incombination therapy with, e.g., chemotherapeutic agents, hormones,antiangiogens, radiolabelled, compounds, or with surgery, cryotherapy,and/or radiotherapy. An antibody of the invention can also beadministered in conjunction with other forms of conventional therapy,either consecutively with, pre- or post-conventional therapy. Forexample, the antibody can be administered with a therapeuticallyeffective dose of chemotherapeutic agent. In another embodiment, theantibody can be administered in conjunction with chemotherapy to enhancethe activity and efficacy of the chemotherapeutic agent. The Physicians'Desk Reference (PDR) discloses dosages of chemotherapeutic agents thathave been used in the treatment of various cancers. The dosing regimentand dosages of these aforementioned chemotherapeutic drugs that aretherapeutically effective will depend on the particular immune disorder,e.g., Hodgkin lymphoma, being treated, the extent of the disease andother factors familiar to the physician of skill in the art and can bedetermined by the physician.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

EXAMPLES Example 1 Materials and Methods used in Examples 1-5

A. Cell Lines

Twenty-one DLBCL cell lines (Ly19, Ly18, Ly10, Ly8, Ly7, Ly4, Ly3, Ly1,Pleiffer, DHL10, DHL8, DHL7, DHL6, DHL5, DHL4, DB, HT WSU, Karpas 422,Toledo and Farage), 1 MLBCL cell line (Karpas 1106) and 7 cHL cell lines(KMH2, HDLM2, SupHD1, L1236, L540, L428, HD-MY-Z) were maintained aspreviously described (Mathas et al. (2002) Embo J 21, 4104-4113; Smithet al. (2005) Blood 105, 308-316). The cHL cell lines were previouslydemonstrated to have constitutive AP-1 activity and increased expressionof c-JUN and JUNB (Mathas et al. (2002) Embo J 21, 4104-4113 and FIG.5).

B. Identification of Genes Overexpressed in cHL Cell Lines by GeneExpression Profiling

Total RNAs from a panel of 21 diffuse large B-cell lymphoma (DLBCL) and7 cHL cell lines were hybridized to U133A and B Affymetrixoligonucleotide microarrays, and the chips were scanned and dataanalyzed as previously described (Monti et al. (2005) Blood 105,1851-1861). The top 9,586 genes that met threshold and variation indexcriteria were analyzed with GenePattern program available on the WorldWide Web at broad.mit.edu/cancer/software/genepattern/CEF) to identifydifferentially expressed genes in cHL and DLBCL. Genes correlated withthe class template (HL vs. DLBCL) were identified by ranking themaccording to their signal-to-noise ratio (SNR). For each gene, aspecific p-value based on permutation testing was calculated andcorrected for false discovery rate by the Benjamini and Hochbergprocedure (Benjamini et al. (2001) Behav Brain Res 125, 279-284; Reineret al. (2003) Bioinformatics 19, 368-375).

C. Analysis of Gal1 Expression in Cell Lines by Immunoblot

DLBCL and cHL cell lines were maintained as previously described (Mathaset al. (2002) Embo J 21, 4104-4113; Polo et al. (2007) Proc Natl AcadSci USA 104, 3207-3212). Cells were lysed, size-fractionated on NuPAGENovex 4-12% Bis-Tris Gels (Invitrogen, Carlsbad, Calif.), andtransferred to PVDF membranes (Millipore Corp., Bedford, Mass.).Membranes were immunostained with purified Gal1 rabbit IgG (Rubinsteinet al. (2004) Cancer Cell 5, 241-251) and HRP-conjugated donkeyanti-rabbit antibody (GE Healthcare, Piscataway, N.J.) and developedusing a chemiluminescent method (ECL, GE Healthcare).

D. Immunohistochemistry

Immunohistochemistry was performed as previously described (Juszczynskiet al. (2006) Mol Cell Biol 26, 5348-5359) using 5 μm thickformalin-fixed, paraffin-embedded tissue sections of newly diagnosedprimary cHLs and DLBCLs and purified Gal1 rabbit IgG.

E. Analysis of Regulatory Elements in the Gal1 Locus and Generation ofGal1 Enhancer Constructs

Computational analysis of the Gal1 locus (chr22:36,400,510-36,406,802,alignment with Human NCBI Genome assembly v36, March 2006) was performedwith the publicly available version of Genomatix suite available on theWorld Wide Web at genomatix.de) (Scherf et al. (2000) J Mol Biol 297,599-606) and rVISTA available on the World Wide Web atrvista.dcode.org/) (Loots and Ovcharenko (2004) Nucleic Acids Res 32,W217-221) and a putative downstream regulatory element (enhancer)containing a conserved AP1 binding site was identified (+1567 to +1675).To generate a series of Gal1 promoter-enhancer reporter constructs, theGal1 promoter region (−403+67) was amplified using PCR and this sequencewas ligated into the pGL3 promoterless reporter vector (Promega,Madison, Wis.), generating pGL3-Gal1-403+67-Luc. Thereafter, fragmentsspanning nucleotides +459+1746, +459+777, and +1346+1746 from the Gal1transcription start site (TSS) were PCR-amplified and cloned intopGL3-Gal1-403+67-Luc 3′ of the luciferase gene. Deletions in AP1 site(TGACTCA to TGxxxCA) were generated using thepGL3-Gal1-403+67-Luc-e1346+1746 construct and the GeneTailorSite-Directed Mutagenesis System (Invitrogen) as recommended by themanufacturer. An additional set of constructs was generated with thecandidate enhancer elements cloned upstream of the Gal1 promoter.

F. Generation of Dominant Negative cJun Constructs

Dominant-negative cJun constructs were generated as previously described(Ludes-Meyers et al. (2001) Oncogene 20, 2771-2780) with minormodifications. A cJun fragment which lacked the transactivation domain(amino acids 123 to 223) was PCR-amplified from intronless cJUN genomicDNA and ligated in the pFLAG-CMV2 vector (pFLAG-CMV2-cJUNDN) (SigmaAldrich, St Louis, Mo.) using forward primer,CAAGAATTCCCAGAACACGCTGCCCAGCGTC (SEQ ID NO: 4), and reverse primer,GAATCTAGAGTCGCAACTTGTCAAGTTCTCAAGTCTGTC (SEQ ID NO: 5).

G. Analysis of Gal1 Promoter—Enhancer Constructs with Luciferase Assays

The HD-MY-Z cHL and SU-DHL7 DLBCL cell lines were grown to 60-80%confluency on 24 well-plates and cotransfected with 300 ng/well of theappropriate promoter-enhancer pGL3 construct (wild-type or mutant Gal1)and 100 ng/well of the control reporter plasmid, pRL-TK (Promega) usingFuGENE 6 transfection reagent (Roche Applied Science) according to themanufacturer's protocol. For cotransfection experiments withcJUN-DN-FLAG, HD-MY-Z cells were transfected with 150 ng ofpGL3-Gal1-403+67-Luc-e1346+1746, 250 ng of pFLAG-CMV2-cJUN-DN and 100 ngof pRL-TK. After 24 hours of incubation, cells were lysed and luciferaseactivities were determined by a chemiluminescence assay using the DualLuciferase Assay kit (Promega) and Luminoskan Ascent luminometer (ThermoLab Systems, Franklin, Mass.) as described (Juszczynski et al. (2006)Mol Cell Biol 26, 5348-5359).

H. Electrophoretic Mobility Shift Analyses of the AP1-Binding Site inthe Gal1 Enhancer

Nuclear extracts from three cHL cell lines (HD-MY-Z, L428 and SupHD-1)and 3 DLBCL cell lines (SU-DHL7, SU-DHL4 and Toledo) were obtained aspreviously described (Juszczynski et al. (2006) Mol Cell Biol 26,5348-5359). Double-stranded wild-type (WT) and mutant probescorresponding to AP1-binding region in Gal1 enhancer (wild-type, WT[5′-TTTTCTGGGTGACTCACTTCCCCCG-3′] (SEQ ID NO: 6) and mutant, MUT[5′-TTTTCTGGGTtcagtACTTCCCCCG-3′ (SEQ ID NO: 7) [mutant bases in lowercase]) were end-labelled with [γ-³²P]ATP, purified and used in bindingreactions as described (Juszczynski et al. (2006) Mol Cell Biol 26,5348-5359). DNA binding was carried out using 5 μg of nuclear extractsand approximately 10,000 cpm of radiolabelled probe in 20 μL of bindingbuffer (Juszczynski et al. (2006) Mol Cell Biol 26, 5348-5359). After 30minutes of incubation, reactions were loaded on a 5% polyacrylamide geland electrophoresed. Gels were vacuum dried and exposed to x-ray filmsovernight at −80° C. For competitor studies, 100× molar excess ofunlabelled wild-type or mutant probe was included in the bindingreactions. For supershift studies, 1 μL of c-JUN antibody or β-actin(Santa Cruz Biotechnology, Santa Cruz, Calif. and Sigma-Aldrich,respectively) was added to the reaction 15 min prior to the probe.

I. Q-PCR Analysis of Gal1 Transcript Abundance Following AP1 Inhibition

The HD-MY-Z cHL cell line was grown to 60-80% confluency on 100 mmplates and transiently transfected with 15 μg of pFLAG-CMV2 (emptyvector) or pFLAGCMV2-cJUNDN plasmids using FuGENE 6 transfection reagent(Roche Applied Science) according to the manufacturer's protocol. After72 hours of culture, RNA was extracted using Trizol reagent (Invitrogen)and cDNA was synthesized from total RNA (3 μg) using SuperScript IIreverse transcriptase (Invitrogen) and random hexamer primers. Gal1 andGAPDH (housekeeping control) transcript abundance was evaluated by QPCRusing Power SYBR green PCR Master Mix (Applied Biosystems, Foster City,Calif.) and the following primers: GAPDH, Forward: GATTCCACCCATGGCAAATTC(SEQ ID NO: 8); GAPDH, Reverse: TGATTTTGG AGGGATCTCGCTC (SEQ ID NO: 9);Gal1, Forward: TCGCCAGCAACCTGAATCTC (SEQ ID NO: 10), Gal1, Reverse:GCACGAAGCTCTTAGCGTCA (SEQ ID NO: 11). PCR was performed using an ABI7700 thermal cycler (Applied Biosystems) and threshold Cycle (C_(T))values were generated using the Sequence Detection Software, version 1.2(Applied Biosystems). Gal1 transcript abundance was calculated relativeto the housekeeping control GAPDH using the 2^(−(ΔCTGal1-ΔCTGAPDH))method according to the manufacturer's instructions. Standard deviationswere calculated from triplicate ΔCT values.

J. RNA-Interference Mediated Gal1 Knock-Down

Gal1 specific siRNA was designed using siRNA Selection Program (Yuan etal. (2004) Nucleic Acids Res 32, W130-134) (available on the World WideWeb at jura.wi.mit.edu/bioc/siRNAext/), synthesized as single-strandedDNA oligonucleotides by Integrated DNA Technologies (IDT, Inc.,Coralville, Iowa) and annealed. Gal1-specific oligonucleotide (Gal1RNAi, GATCCGCTGCCAGATGGATACGAATTCAAGAGATTCGTATCCATCTGGCAGCTTTTTTG (SEQID NO: 12) or scrambled oligonucleotide (SCR,GATCCCCTCCATATCTCGCGCGTCTTCAAGAGAGACGCGCGAGATATGGAGGTTTTTTG (SEQ ID NO:13) were ligated into the linearized pSIREN-RetroQ retroviral vector (BDClontech, Mountain View, Calif.). Generation of recombinant retrovirusand infection of HD-MY-Z cells was performed as previously described(Juszczynski et al. (2006) Mol Cell Biol 26, 5348-5359). Afterinfection, cells were subjected to puromycin selection (0.5 μg/mL) andsubcloning by limiting dilution. Thereafter, whole-cell extracts ofobtained subclones were prepared and screened for Gal1 expression byimmunoblotting as described above. Gal1 knockdown did not alter theproliferation rate or viability of transduced HD-MY-Z cells.

K. Co-Cultures and Analyses of T-Cell Responses in cHL Microenvironment

1. Co-culture. Peripheral blood mononuclear cells (PBMCs) were obtainedfrom normal blood donors by Ficoll-Hypaque (GE Healthcare) gradientcentrifugation. Thereafter, T cells were purified using Pan T CellIsolation Kit II (Miltenyi Biotec, Auburn, Calif.) and activated with 1μg/mL PHA (Sigma-Aldrich) for 64 hours. 1×10⁶ of activated T cells werethen co-cultured with monolayers (3×10⁶ cells) of HD-MY-Z cellsexpressing either scrambled shRNA or Gal1 specific shRNA for 6 hours at37° C.

2. Analyses of viable T cells. Following co-culture, all cells wereharvested and sequentially stained with CD3-PE and CD4-PE-Cy5 (BeckmanCoulter, Fullerton, Calif.) followed by Annexin V-FITC and analyzed witha Beckman Cytomics FC500 flow cytometer. The numbers of viable (annexinV-) and (propidium iodide-) CD3⁺ and CD4⁺ T cells in Gal1 shRNA orscrambled shRNA HD-MY-Z/T-cell co-cultures were then compared.

3. Analyses of T-bet and GATA-3 expression in co-cultured CD4⁺ T cells.After PHA-activated T-cells were co-cultured with HD-MY-Z cellsexpressing either scrambled shRNA or Gal1 specific shRNA for 24-48hours, cells were collected and non-viable cells were depleted byFicoll-Hypaque gradient centrifugation. Remaining viable cells werewashed, incubated with CD4 MACS microbeads and purified using a MACS LScolumn according to the manufacturer's protocol (Miltenyi Biotec).Thereafter, RNA was obtained from the isolated CD4⁺ cells and cDNA wassynthesized from total RNA (3 μg) as described above. GATA-3, T-bet andGAPDH (housekeeping control) transcript abundance was evaluated by QPCRusing Power SYBR green PCR Master Mix (Applied Biosystems, Foster City,Calif.), GAPDH primers listed above and the following GATA3 and T-betprimers: GATA3, Forward: TAACATCGACGGTCAAGGCA (SEQ ID NO: 14); GATA3,Reverse: ACACCTGGCTCCCGTGGT (SEQ ID NO: 15); T-bet, Forward:TGGACGTGGTCTTGGTGGACC (SEQ ID NO: 16); T-bet, Reverse:TGGACGTACAGGCGGTTTCC (SEQ ID NO: 17). PCR and transcript abundanceanalysis was performed as described above. GATA-3 and T-bet expressionin purified CD4⁺ cells from SCR shRNA and Gal1 shRNA HD-MY-Z/T-cellcocultures were then compared.

L. Cytokine Production in T-Cell Subpopulations Treated with rGal1

Recombinant Gal1 was obtained and purified essentially as described(Toscano et al. (2006) J Immunol 176, 6323-6332). T cells were purifiedas described above and simultaneously activated with anti-CD3-(0.1μg/mL) and anti-CD28-(0.5 μg/mL) coated latex beads (Invitrogen) andtreated with rGal1 (20 μM) in RPMI 1640 medium containing 10% fetalbovine serum and 1 mM β-ME alone or rGal1 and the Gal1 inhibitor,thiodigalactoside (TDG, 100 mM) (Rabinovich et al. (1999) J Exp Med 190,385-397) or left untreated for 16 hours. Supernatants were then analyzedfor IL-4, IL-5, IL-10, and IL-13 using cytometric bead array (CBA) FlexSet beads according to manufacturer's protocol (BD Biosciences, SanJose, Calif.). In brief, multiplexed antibody-conjugated beads wereincubated with culture supernatants or serial dilutions of cytokinestandards for 1 hour. Thereafter, the PE detection reagent was added andsamples were incubated for an additional 2 hours, washed and analyzedusing FACS Aria Flow Cytometer (BD Biosciences). Results were capturedwith FACS Diva software and analyzed with the FCAP1.1 program (BDBiosciences).

M. Analyses of Regulatory T Cells (T_(regs))

T cells were purified, activated with anti-CD3- and anti-CD28-coatedlatex beads and treated with rGal1, rGal1 and TDG or left untreated for24 hours. Thereafter, cells were stained with CD4-FITC and CD25-APC,washed, fixed, permeabilized and stained with FOXP3-PE antibody or ratIgG2b isotype control as previously described (Zorn et al. (2006) Blood108, 1571-1579) (Human Regulatory T-cell staining kit, eBioscience, SanDiego, Calif.). CD4⁺ CD25^(high) FOXP3 T_(reg) cells were identified andquantified using Beckman Cytomics FC500 flow cytometer as previouslydescribed (Zorn et al. (2006) Blood 108, 1571-1579).

N. Statistical Analysis

All statistical analyses were done using Statistica 6.0 software(Statistica, Tulsa, Okla.). Students t test was used for comparisonsbetween 2 groups; Anova was used for multiple comparisons.

Example 2 Overexpression of Gal1 in cHL RS Cells

To identify novel cHL-specific T-cell inhibitory molecules, the geneexpression profiles of a series of cHL and diffuse large B-cell lymphoma(DLBCL) and mediastinal LBCL (MLBCL) cell lines were compared. Gal1transcripts were 4- to 29-fold more abundant in cHL cell lines than inthe LBCL lines (p=0.002, FDR=0.014, FIGS. 1A and 1B). Gal1 proteinexpression was also uniformly high in cHL cell lines and low orundetectable in DLBCL and MLBCL lines by western blotting (FIG. 1C).Immunohistochemical staining of primary tumor sections revealed abundantGal1 expression in cHL RS cells, whereas LBCLs were uniformly negative(FIG. 1D). In a series of primary lymphoid tumors, 10/10 cHLs were Gal1⁺whereas 10/10 primary DLBCLs and 5/5 primary mediastinal LBCLs (MLBCLs)lacked Gal1 expression. Taken together, these data indicate that Gal1 isselectively upregulated in the RS cells of cHLs.

Example 3 RS Cell Gal1 Expression is Regulated by an AP1-DependentEnhancer

To elucidate the mechanisms responsible for Gal1 overexpression in cHLRS cells, the Gal1 locus on chromosome 22 was analyzed. A candidateGC-rich regulatory element with a conserved putative AP1 binding siteapproximately 1.5 kb downstream of the Gal1 transcription start site(TSS) was identified. Since the AP1 components, cJUN and JUN-B areoverexpressed in cHL and are critical for the pathogenesis of thedisease (Mathas et al. (2002) EMBO J. 21: 4104-4113), it was askedwhether AP1 mediates Gal1 expression in cHL. Luciferase vectors drivenby the previously described Gal1 promoter (−403+67) were generated(Salvatore et al. (1998) FEBS Lett 421:152-8) and the putative Gal1enhancer element (or mutated controls) and assessed associatedluciferase activity in a cHL cell line (HD-MY-Z) known to haveconstitutive activation of AP1 (FIG. 2A) (Mathas et al. (2002) EMBO J.21: 4104-4113). Constructs including the GC-rich regulatory element(bp+459+1746 or +1346+1746) upregulated luciferase expression 8-10 fold,whereas constructs lacking the candidate sequence (+459+777) orcontaining a deletion in the AP1-binding site (+1346+1746_(del))exhibited significantly lower luciferase activity (FIG. 2A). Similarresults were obtained with a set of constructs in which the regulatoryelement was cloned upstream of the Gal1 promoter, demonstrating that theidentified sequence (bp+1346+1746) is a bona fide Gal1 enhancer.

Given the AP1 dependence of the Gal1 enhancer and the constitutive AP1activity in cHL (Mathas et al. (2002) EMBO J. 21: 4104-4113), it wasnext asked whether the Gal1 enhancer was selectively active in thisdisease. For these experiments, cHL, DLBCL and fibroblast cell lineswere transfected with either the Gal1 promoter-only vector(pGL3-Gal1₄₀₃₊₆₇-Luc) or the promoter-enhancer construct(pGL3-Gal1₄₀₃₊₆₇-Luc-e₁₃₄₆₊₁₇₄₆) and compared the respective luciferaseactivities (FIG. 2B). The Gal1 promoter-enhancer construct specificallyupregulated luciferase expression in cHL cells but not DLBCL cells orfibroblasts (FIG. 2B).

After demonstrating the specificity and activity of the Gal1 AP1enhancer element in a cHL cell line (FIGS. 2A and 2B), the requirementfor AP1 transcription factors in electrophoretic mobility shift assayswas directly evaluated (FIG. 2C). Nuclear extracts from 3 cHL and 3DLBCL cell lines were incubated with radiolabelled wild-type or mutantprobes corresponding to an AP1 element in the Gal1 enhancer. Gal1 wildtype, but not mutant probe, directly bound to nuclear proteins extractedfrom cHL, but not DLBCL, cell lines (FIG. 2C). The complexes formed withGal1 WT probe were displaced by unlabeled WT competitor, furtherconfirming the binding specificity (FIG. 2C). In supershift assays, theGal1/AP1 complex was retarded by cJun antibody (FIG. 2C). Furthermore,the simultaneous overexpression of a dominant negative cJUN construct(cJUN-DN) reduced Gal1-driven luciferase activity in cHL cells (FIG.2D). In addition, when AP1 was at least partially inhibited via theoverexpression of cJUN-DN, there was a significant decrease in Gal1transcript abundance in cHL cells (FIG. 2E). Taken together, thesestudies indicate that cHL RS cells selectively overexpress Gal1, atleast in part, via an AP1-driven enhancer.

Example 4 Endogenous cHL Gal1 Expression Contributes to theImmunosuppressive and Th2-Skewed Microenvironment in Classical HodgkinLymphoma

After delineating the mechanism for cHL-specific overexpression of Gal1,the functional consequences of cHL RS cell Gal1 expression on theassociated inflammatory/immune infiltrate was assessed. Stable HD-MY-Ztransfectants expressing Gal1 specific shRNA (Gal1 shRNA) or a scrambledcontrol shRNA (SCR, FIG. 3A) were generated. Activated T-cell blastswere then added to Gal1 shRNA or SCR HD-MY-Z cells grown as adherentmonolayers and the cHL line and T cells were co-cultured. Thereafter,total T-cell and Th-cell viabilities were assessed using 3-colorAnnexin-V, -CD3 and -CD4 flow cytometry. There were significantly fewerviable total (CD3⁺) and Th (CD4⁺) T cells in control (SCR) HD-MY-Zco-cultures than in co-cultures of HD-MY-Z cells with Gal1 knockdown(FIG. 3B). These studies directly demonstrate that endogenous cHL RScell Gal1 decreases the viability of infiltrating activated T cells.

Given the skewed nature of inflammatory infiltrate in cHL, it was askedwhether endogenous cHL RS cell Gal1 may contribute to this Th1/Th2imbalance. To address this question, CD4⁺ Th cells were isolated fromthe cHL/T-cell co-cultures and the relative expression of the Th1- andTh2-specific transcription factors, T-bet and GATA-3, was analyzed byRQ-PCR. CD4⁺ Th cells co-cultured with control (SCR) HD-MY-Z cellsexhibited significantly lower expression of T-bet and higher expressionof GATA-3 than CD4⁺ T cells from Gal1 shRNA HD-MY-Z co-cultures (FIG.3C). Taken together, these results indicate that endogenous cHL RS cellGal1 selectively decreases the viability of associated Th1 cellsresulting in a skewed Th2-type infiltrate.

Example 5 Gal1 Promotes Immune Privilege by Favoring the Secretion ofTh2 Cytokines and the Expansion of CD4⁺ CD25^(high) FOX3⁺ T_(regs)

Given the profound immunosuppressive activity of Gal1 inTh1/Th17-mediated autoimmune settings (Rabinovich et al. (1999) J ExpMed 190:385-397; Toscano et al. (2006) J Immunol 176:6323-6332; Santucciet al. (2003) Gastroenterol 124: 1381-1394), it was asked whether thisglycan-binding protein was directly implicated in the skewed Th2cytokine profile associated with primary cHL. For this purpose,activated T cells were treated with recombinant Gal1 (rGal1) in thepresence or absence of the Gal1 inhibitor, thiodigalactoside (TDG)(Rabinovich et al. (1999) J Exp Med 190:385-397). As expected, rGal1induced apoptosis of total activated T cells (FIG. 4). Concurrenttreatment with TDG completely blocked rGal1-induced apoptosis,confirming the specificity of the Gal1 effect (FIG. 4). Th2 cytokines insupernatants from activated T cells that were untreated or treated withrGal1 in the presence or absence of TDG were subsequently quantified.Supernatants from rGal1-treated T cells contained significantly higheramounts of the Hodgkin-associated Th2 cytokines, IL-4, IL-5, IL-10 andIL-13, and TDG specifically blocked this effect (FIG. 3D). These datafurther support the hypothesis that RS cell Gal1 expression promotesTh2-type cytokine production in primary cHLs.

In addition to Th2 cells, the inflammatory infiltrate in primary cHLincludes abundant T regulatory cells (CD4⁺ CD25^(high) FOXP3⁺) thatdirectly blunt the host anti-tumor immune response (Marshall et al.(2004) Blood 103:1755-1762; Gandhi et al. (2006) Blood 108:2280-2289,Ishida et al. (2006) Cancer Res 66:5716-5722; Gabrilovich, D. (2007)Curr Cancer Drug Targets 7: 1). Therefore, the role of Gal1 in theselective expansion of T_(reg) cells was assessed using theabove-mentioned assay. Activated T cells were treated with rGal1 in thepresence or absence of TDG and analyzed thereafter for T_(regs) (CD4⁺CD25^(high) FOXP3⁺) using triple-color immunofluorescence as described(Zorn et al. 2006) Blood 108:1571-1579). The CD4⁺ CD25 high FOXP3population was significantly increased in rGal1 treated cells and TDGcompletely blocked this effect (FIG. 3E). Taken together, these resultsdemonstrate that Gal1 fosters the skewed and immunosuppressivemicroenvironment in cHL by enhancing the production of Th2 cytokines(IL-4, IL-5, IL-10 and IL-13) and increasing the relative abundance ofT_(reg) cells.

Examples 1-5 describe the overexpression of Gal1 by cHL RS cells andidentify the mechanism as a phylogenetically conserved AP1 responsiveenhancer. In functional in vitro assays, it has been shown that cHL RScell Gal1 decreased the viability of activated T cells and skewed thebalance towards a Th2 immune response. Consistent with this observation,Gal1 markedly increased the secretion of Th2 cytokines including IL-4,IL-5, IL-10 and IL-13. In addition, Gal1 fostered the expansion and/orretention of CD4⁺ CD25^(high) FOXP3⁺ T_(reg) cells. Taken together,these data directly implicate RS cell Gal1 in the development andmaintenance of the unique Th2/T_(reg)-skewed immunosuppressivemicroenvironment in primary cHL.

Although cHL RS cells exhibit near uniform Gal1 expression, DLBCLs andMLBCL are largely Gal1 negative, prompting speculation that Gal1 maydistinguish cHL from certain “grey zone” lymphomas that sharecharacteristics of DLBCL and cHL (Abramson and Shipp (2005) Blood 106,1164-1174). A common feature of these “grey zone” lymphomas is anincreased host inflammatory response, highlighting the interactionbetween the tumor cells and their host microenvironment (Abramson andShipp (2005) Blood 106, 1164-1174). Gal1 overexpression is a definingfeature of cHL that is not shared with its closely related counterpart,primary MLBCL (Savage et al. (2003) Blood 102, 3871-3879), providinginsights into the relative efficacy of host immune responses in thesetumors.

The differential expression of Gal1 in these lymphomas is likely due tothe cHL-specific overexpression of the AP1 transcription factorcomponents, cJUN and JUNB, and the constitutive activation of the AP1pathway (Mathas et al. (2002) Embo J 21, 4104-4113). Gal1 expression isregulated, at least in part, by a cHL-specific, AP1-driven enhancer. AP1also functions in synergy with NF-κB to control the proliferation andlimit the apoptosis of cHL RS cells (Mathas et al. (2002) Embo J 21,4104-4113). Therefore, in addition to its pro-survival functions in cHLRS cells, AP1 also regulates the interplay between RS cells and thetumor microenvironment through a Gal1-mediated pathway.

RS Gal1 is likely to be a critical factor shaping the immuno- andhistopathologic features of primary cHL. Endogenous RS cell Gal1specifically induced the apoptosis of activated T lymphocytes,suggesting that a similar mechanism operates in primary cHLs and thatthese tumors represent sites of immune privilege. Gandhi et al. alsorecently described increased Gal1 expression in cHL (Gandhi et al.(2007) Blood, First Edition Paper online Apr. 16, 2007).

The short-term in vitro assays likely underestimate the long-term invivo effects of Gal1 in cHL because the lectin is also deposited in theextracellular matrix and stroma where it kills susceptible T cells (Heand Baum (2004) J Biol Chem 279, 4705-4712). In in vitro assays, Gal1expressing RS cells selectively decreased the viability of infiltratingTh1 cells resulting in a Th2-predominant infiltrate. Consistent with theobserved reduction in Th1 cells and enrichment in Th2 cells, Gal1significantly increased the levels of the Th2 cytokines, IL-4, IL-5,IL-10 and IL-13. The Gal1-associated cytokine profile suggests that thisglycan-binding protein may have additional functions beyond modulatingT-cell responses in primary cHLs. Since IL-13 is a critical RS cellgrowth factor (Skinnider et al. (2002) Leuk Lymphoma 43, 1203-1210),Gal1 may indirectly stimulate tumor growth by fostering the productionof this Th2 cytokine. Via its effect on another Th2 cytokine, IL-5, Gal1may also promote the characteristic eosinophilic infiltrate in primarycHL (von Wasielewski et al. (2000) Blood 95, 1207-1213).

The observations regarding Gal1 function in cHL are consistent withrecent reports regarding the role of the lectin in murine models ofTh1-driven chronic inflammatory and autoimmune disorders includingcollagen-induced arthritis, inflammatory bowel disease, graft vs. hostdisease and autoimmune uveitis (Rabinovich et al. (1999) J Exp Med190:385-397; Toscano et al. (2006) J Immunol 176:6323-6332; Santucci etal. (2003) Gastroenterol 124: 1381-1394; Baum et al. (2003) Clin Immunol109:295-307). In these studies, the administration of Gal1 dramaticallysuppressed Th1-dependent responses and skewed towards Th2 cytokineprofiles (Rabinovich et al. (1999) J Exp Med 190:385-397; Toscano et al.(2006) J Immunol 176:6323-6332; Santucci et al. (2003) Gastroenterol124: 1381-1394; Baum et al. (2003) Clin Immunol 109:295-307; van derLeig et al. (2006) Mol Immunol 10, 1-8). A careful examination of themechanisms involved in Gal1-mediated Th2-skewing recently revealed thatTh1 cells express the repertoire of cell surface glycans required forGal1 binding and subsequent cell death, whereas Th2 cells are protectedfrom Gal1 via differential sialylation of their cell surfaceglycoproteins (Toscano et al. (2007) Nature Immunol, Published online:24 Jun. 2007).

In addition to the Th2 shift, the results provide the first evidenceshowing that Gal1 treatment increases the relative abundance of CD4⁺CD25^(high) FOXP3 T_(reg) cells that may blunt the host anti-cHL immuneresponse. It is possible that the specific glycosylation pattern of Gal1receptors on T_(regs) renders them resistant to Gal1-induced apoptosis.In fact, T_(regs) have been reported to exhibit increased α2,6sialylation (compared to effector T cells) (Jenner et al. (2006) ExpHematol 34, 1211-1217); this selective sialylation might interfere withGal1 binding and cell death (Amano et al. (2003) J Biol Chem 278,7469-7475). This hypothesis is further supported by recent studiesdemonstrating that T_(regs) overexpress Gal1 and remain resistant toGal1-mediated apoptosis (Garin et al. (2007) Blood 109, 2058-2065).

Taken together, the data described herein provide new insights into thebiology of RS cells and identifies a key AP1-dependent mechanismregulating cHL-specific immune privilege. Since Gal1 blockadedramatically increased tumor rejection in recently described murinemodels (Rubinstein et al. (2004) Cancer Cell 5, 241-251), it is possiblethat Gal1 inhibition may augment host anti-tumor responses in primarycHL. Furthermore, this lectin is likely to have additional roles in thebiology of cHL. Recent studies indicate that Gal1 also promotes tumorcell motility and enhances tumor angiogenesis (Liu and Rabinovish,(2005) Nature Reviews Cancer 5, 29-41; Rabinovich (2005) Br J Cancer 92,1188-1192; Thijssen et al. (2006) Proc Natl Acad Sci USA 103,15975-15980; Camby et al. (2002) J Neuropathol Exp Neurol 61, 585-596),processes critical for tumors like cHL that spread by contiguousinvolvement of adjacent nodes and organs. Gal1, thus, represents a newrational therapeutic target in cHL.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) on the world wide web attigr.org and/or the National Center for Biotechnology Information (NCBI)on the world wide web at ncbi.nlm.nih.gov.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for modulating an immune responsecomprising contacting an immune cell from a subject having Hodgkinlymphoma in vitro with an agent that modulates the interaction betweengalectin-1 (Gal1) or a fragment thereof and its natural bindingpartner(s), wherein the agent is a blocking antibody, or anantigen-binding fragment thereof, that recognizes Gal1, and wherein theagent does not comprise a Hodgkin lymphoma-specific antigen, anantigen-binding molecule thereof, or an immune cell obtained from thesubject, to thereby modulate the immune response.
 2. The method of claim1, wherein the immune response is upregulated.
 3. The method of claim 1,further comprising contacting the immune cell with an additional agentthat upregulates an immune response.
 4. A method for treating a subjecthaving Hodgkin lymphoma comprising administering an agent that inhibitsthe interaction between galectin-1 (Gal1) and its natural bindingpartner(s) on cells of the subject, wherein the agent is a blockingantibody, or an antigen-binding fragment thereof, that recognizes Gal1,and wherein the agent does not comprise a Hodgkin lymphoma-specificantigen, an antigen-binding molecule thereof, or an immune cell obtainedfrom the subject, such that the Hodgkin lymphoma is treated.
 5. Themethod of claim 4, further comprising administering a second agent thatupregulates an immune response in the subject.
 6. The method of claim 4further comprising a chemotherapy treatment.